Use of foti to enhance stem cell mobilization and proliferation

ABSTRACT

The present invention provides a method of using  Polygonum multiflorum  or extracts thereof to enhance stem cell mobilization in a subject, including hematopoietic stem cells (HSCs) and bone marrow stem cells (BM-SCs). In one embodiment, a blended composition of plant parts, fruits, mushrooms, microorganisms, maternal fluids, and extracts thereof are used to promote mobilization of stem cells, resulting in migration of the stem cells to specific sties of maintenance and repair within tissues and/or organs. Increased circulation of HSCs and/or BMSCs and migration towards sites of maintenance promotes the natural regeneration mechanisms in the body.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a National Phase of International Application No.PCT/US2012/065294, filed Nov. 15, 2012, which designated the U.S. andthat International Application was published under PCT Article 21(2) inEnglish. This application also includes a claim of priority under 35U.S.C. 119(e) to U.S. provisional patent application No. 61/561,431,filed Nov. 18, 2011.

FIELD OF THE INVENTION

The present invention relates to methods and compositions for enhancingthe mobilization of stem cells.

BACKGROUND OF THE INVENTION

All publications herein are incorporated by reference to the same extentas if each individual publication or patent application was specificallyand individually indicated to be incorporated by reference. Thefollowing description includes information that may be useful inunderstanding the present invention. It is not an admission that any ofthe information provided herein is prior art or relevant to thepresently claimed invention, or that any publication specifically orimplicitly referenced is prior art.

Stem cells (SCs) are defined as cells with the unique capacity toself-replicate throughout the entire life of an organism and todifferentiate into various cell types of the body. Two well-known typesof stem cells are embryonic stem cells and adult stem cells. Embryonicstem cells (ESCs) are extracted from 5-10 day old embryos calledblastulas. Once isolated, ESCs can be grown in vitro and led todifferentiate into various types of tissue cell (such as heart cells,liver cells, nervous cells, and kidney cells), after which they can beinjected in specific tissues in order to regenerate the tissue.

Adult stem cells (ASCs) are undifferentiated or primitive cells that canself-renew and differentiate into specialized cells of various tissuesand are found in any living organism after birth. ASCs have beenisolated from various tissues such as the liver (oval cells) (Wang etal., 2003), the intestine (intestinal crypt stem cells) (Barker et al.,2008), muscles (satellite cells) (Kuang et al., 2008), the brain (neuralstem cells) (Revishchin et al., 2008), and recently the pancreas (nestinpositive pancreatic stem cells) (Burke et al., 2007). Umbilical cordstem cells and placental stem cells are considered ASCs.

The role of ASCs found in tissues (tissue stem cells) is to maintain andrepair the tissue in which they are found, although recent studies havereported that ASCs from one tissue may have the ability to develop intocell types characteristic of other tissues. For example, oval cells inthe liver were shown in vitro to have the ability to becomeinsulin-producing pancreatic cells. (Yang et al. 2002) Nevertheless, thegeneral view is that local stem cells are primarily involved in minorrepair of the tissue in which they reside. In the case of significantinjury or degeneration, the number of new tissue cells found in healingtissue far exceeds the capacity of local stem cells to duplicate anddifferentiate, suggesting that stem cells coming from other sites mustbe involved in the process of repair.

Although many tissues contain their own specific population of tissuestem cells, certain ASCs of key interest are those primarily found inthe bone marrow and blood, Tissue stem cells are traditionally believedto be limited in their ability to differentiate into other tissues,however bone marrow stem cells (BMSC) were recently shown to havesignificant capability to become cells of other tissues.

It is difficult to freeze these processes in time to extract a cohesive,comprehensive portrait of regenerative mechanisms in the body.Nonetheless, enough information is available to affirm that differentstem cells in the body, whether BMSCs, HSCs, marrow stromal cells(MSCs), multipotent adult progenitor cells (MAPCs), very smallembryonic-like stem cells (VSEL), epiblast-like stem cell (ELSC) orblastomere-like stem cell (BLSC), constitute a broad component of thebody's natural healing system. Since stem cells are capable ofdifferentiating into a broad variety of cell types, they play animportant role in the healing and regenerative processes of varioustissues and organs. Bone marrow stem cells, including marrow stromalcells (MSCs), are released from tissues of origin, and circulate in asubject's circulatory or immune system to migrate into various organsand tissues to become mature, terminally differentiated cells.Therefore, enhancement of stem cell trafficking (i.e., release,circulation, homing and/or migration) can amplify these physiologicalprocesses and provide potential therapies for various pathologies. Thereare compositions and methods that utilize stem cell mobilization as atherapeutic approach. However, existing methods of promoting stem cellmobilization suffer from significant drawbacks, including poor kineticperformance, high cost, inconvenient methods of administration andunwanted side effects. One leading approach, injection of granulocytecolony-stimulating factor (G-CSF) or recombinant forms thereof, requiresdays to achieve peak circulating HSC numbers. The opposite problemexists with administration of interleukin-8 (IL-8), which acts onlywithin minutes and has a short-lived effect on elevating circulating HSClevels in the bloodstream. (Frenette et al., 2000; Jensen et al., 2007)G-CSF and a different molecule, CXCR4 antagonist AMD3100, can havesignificant side effects, including hemorrhaging, rupturing of thespleen, bloody sputum, bone disorders, among others. Thus, there is aneed in the art for an effective and convenient method for deliveringstem cell mobilization agents to human subjects, to obtain positiveclinical benefits without side effects and at a reduced cost.

Accordingly, the inventive compositions and methods disclosed hereinenhance the release, circulation, homing and/or migration of stem cellswithin the body to promote healing and treatment of damaged tissues, aswell as aid in the regeneration of tissues that suffer from some levelof cellular loss, for greater vitality and reduced incidence of disease.

SUMMARY OF THE INVENTION

The present invention includes a method of increasing stem cellmobilization in a subject, including providing a mobilization agentcapable of increasing stem cell mobilization, and administering aquantity of the mobilization agent to the subject in an amountsufficient to increase stem cell mobilization in the subject. In adifferent embodiment the mobilization agent is a composition includingone or more of the following components selected from the group thatincludes: Polygonum multiflorum or extracts thereof, Lycium barbarum orextracts thereof, blue-green algae or extracts thereof, colostrum orextracts thereof, spirulina or extracts thereof, fucoidan, Hericiumerinaceus or extracts thereof, Ganoderma Lucidum or extracts thereof,and Cordyceps Sinensis or extracts thereof. In a different embodiment,the mobilization agent is Polygonum multiflorum or extracts thereof. Ina different embodiment, the stem cell is a bone marrow-derived stem cell(BMSC). In a different embodiment, the stem cell is a hematopoietic stemcell (HSC). In a different embodiment, administering the quantitycomprises oral administration. In a different embodiment, the oraladministration comprises use of a capsule or a pill.

Another aspect of the present invention described herein is apharmaceutical composition including one or more of the followingcomponents selected from the group that is: Polygonum multiflorum orextracts thereof, Lycium barbarum or extracts thereof, blue-green algaeor extracts thereof, colostrum or extracts thereof, spirulina orextracts thereof, fucoidan, Hericium erinaceus or extracts thereof,Ganoderma Lucidum or extracts thereof, and/or Cordyceps Sinensis orextracts thereof; and a pharmaceutically acceptable carrier. In adifferent embodiment, the quantity of Polygonum multiflorum or extractsthereof, comprises 10-2000 mg in a single dose. In a differentembodiment, the quantity of Lycium barbarum or extracts thereof,comprises 10-2000 mg in a single dose. In a different embodiment, thequantity of blue-green algae or extracts thereof, comprises 10-2000 mgin a single dose. In a different embodiment, the quantity of colostrumor extracts thereof, comprises 10-300 mg in a single dose. In adifferent embodiment, the quantity of spirulina or extracts thereof,comprises 10-300 mg in a single dose. In a different embodiment, thequantity of fucoidan or extracts thereof, comprises 10-2000 mg in asingle dose. In a different embodiment, the quantity of Hericiumerinaceus or extracts thereof, Ganoderma Lucidum or extracts thereof,and/or Cordyceps Sinensis or extracts thereof comprises 10-1000 mg in asingle dose.

Another aspect of the present invention described herein is method ofpreparing an extract of Polygonum multiflorum including providing aquantity of Polygonum multiflorum, crushing the Polygonum multifloruminto a solid powder, drying the solid powder, immersing the solid powderin a solvent, and separating the solid power from the solvent, whereinthe solvent includes an extract of Polygonum multiflorum. In a differentembodiment, immersing the solid powder is in a solvent that is water oranother aqueous solution at a low temperature. In a differentembodiment, the low temperature is approximately 4° C. In a differentembodiment, immersing the solid powder is in a solvent that is anorganic solvent at a high temperature. In a different embodiment, thehigh temperature is near the boiling point of the organic solvent. In adifferent embodiment, the organic solvent is methanol or ethanol. In adifferent embodiment, the Polygonum multiflorum is frozen beforecrushing.

BRIEF DESCRIPTION OF THE FIGURES

Exemplary embodiments are illustrated in referenced figures. It isintended that the embodiments and figures disclosed herein are to beconsidered illustrative rather than restrictive.

FIG. 1 depicts mobilization and migration of endogenous stem cells inaccordance with various embodiments of the present invention. Undernormal physiological conditions or in response to disease or injury,hematopoietic stem cells mobilize from compartments such as bone (A) andcirculate into the bloodstream (B), migrate towards tissues to promoterepair and regeneration in different parts of the body (C).

FIG. 2 shows a schematic illustration of the steps involved in themigration of a stem cell, underscoring the role of CXCR4, in accordancewith an embodiment of the present invention.

FIG. 3 provides graphs illustrating a typical time course of stem cellmigration in the human body after consumption of (A) whole Lyciumbarbarum (LB) fruit and (B) colostrum (Col), in accordance with variousembodiments of the present invention. For both products, the thin linesshow individual responses. For LB, the thick dotted line is the averageresponse while the thick line shows the time course of the response withthe average peak response at 45 minutes. For Col, all participantspeaked at 60 minutes, so the thick lines show the average time course ofthe response.

FIG. 4 provides a graph illustrating a typical time course of stem cellmigration in the human body after consumption of (A) a polysacchariderich fraction of mushroom (Cordyceps sinensis, Ganoderma lucidum,Hericium erinaceus), and (B) spirulina or an extract thereof, inaccordance with an embodiment of the present invention.

FIGS. 5A, 5B and 5C are flow cytometry profiles of blood samples showingthe proportions of CD34+ lymphocytes from the peripheral blood of ahuman volunteer after ingestion of L. barbarum, colostrum and mushroompolysaccharides, respectively, in accordance with an embodiment of thepresent invention. The X axis displays fluorescence intensity of thestem cell marker. The M1 marker indicates events showing positivity forthe stem cell marker CD34.

FIG. 6 is a graph illustrating the expression of CXCR4 molecules on thesurface of CD34+ circulating stem cells before and after consumption ofLB, Col, and mushroom polysaccharides, in accordance with an embodimentof the present invention.

FIG. 7 provides a graph illustrating a typical time course of stem cellmigration in the human body after consumption of Lycium barbarum,colostrum, spirulina and a polysaccharide rich fraction of mushroom(Cordyceps sinensis, Ganoderma lucidum, Hericium erinaceus), inaccordance with an embodiment of the present invention.

FIG. 8 depicts changes in circulating CD34+ hematopoietic stem cells inhuman volunteers following oral administration of fucoidan extractedfrom Undaria pinnatifida in accordance with various embodiments of thepresent invention. Baseline levels of peripheral blood stem cells werequantified in volunteers. Volunteers then ingested 250 mg of fucoidanextracted from Undaria pinnatifida. The levels of stem cells weresubsequently measured at 45, 90 and 180 minutes. The number ofcirculating stem cells increased on average by 17%, 23% (P<0.02) and 32%(P<0.02), respectively.

FIG. 9 depicts the results of consuming fucoidan from algae species,Chordaria cladosiphon in accordance with various embodiments of thepresent invention. Consumption of 250 mg of fucoidan from Chordariacladosiphon gave an average decrease in the number of circulating stemcells under the same conditions.

FIG. 10 depicts the results of consuming a combination of Polygonummultiflorum, blue-green algae, and fucoidan in accordance with variousembodiments of the present invention. Consumption of the combinationincluding Polygonum multiflorum resulted in a transitory increase in thenumber of circulating stem cells compared to placebo under the sameconditions. The combination containing Polygonum multiflorum was shownto trigger a modest increase in the number of circulating stem cells by13±6% (n=7) (p<0.05). The increase exceeded 25% in 2 of theparticipants.

DETAILED DESCRIPTION OF THE INVENTION

All references cited herein are incorporated by reference in theirentirety as though fully set forth. Unless defined otherwise, technicaland scientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which this inventionbelongs. Singleton et al., Dictionary of Microbiology and MolecularBiology 3^(rd) ed., J. Wiley & Sons (New York, N.Y. 2001); March,Advanced Organic Chemistry Reactions, Mechanisms and Structure 5^(th)ed., J. Wiley & Sons (New York, N.Y. 2001); and Sambrook and Russell,Molecular Cloning: A Laboratory Manual 3rd ed., Cold Spring HarborLaboratory Press (Cold Spring Harbor, N.Y. 2001), Remington'sPharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton,Pa., 15th Edition (1975), describes compositions and formulationssuitable for pharmaceutical delivery of the inventive compositionsdescribed herein provide one skilled in the art with a general guide tomany of the terms used in the present application.

One skilled in the art will recognize many methods and materials similaror equivalent to those described herein, which could be used in thepractice of the present invention. Indeed, the present invention is inno way limited to the methods described herein. For purposes of thepresent invention, the following terms are defined below.

“Administering” and/or “administer” as used herein refer to any routefor delivering a pharmaceutical composition to a patient. Routes ofdelivery may include non-invasive peroral (through the mouth), topical(skin), transmucosal (nasal, buccal/sublingual, vaginal, ocular andrectal) and inhalation routes, as well as parenteral routes, and othermethods known in the art. Parenteral refers to a route of delivery thatis generally associated with injection, including intraorbital,infusion, intraarterial, intracarotid, intracapsular, intracardiac,intradermal, intramuscular, intraperitoneal, intrapulmonary,intraspinal, intrasternal, intrathecal, intrauterine, intravenous,subarachnoid, subcapsular, subcutaneous, transmucosal, or transtracheal.Via the parenteral route, the compositions may be in the form ofsolutions or suspensions for infusion or for injection, or aslyophilized powders.

“Blue-green algae” as used herein refers to the common name forgram-negative photosynthetic bacteria belonging to division Cyanophytathat may exist in unicellular, colonial, or filamentous forms.Representative blue-green algae include, but are not limited to,Spirulina and Aphanizomenon species, one example being the Aphanizomenonflos aquae (AFA) species of blue-green algae. “Algae” is the plural formof “alga,” which is a cell of a microalgae species. For example, “bluegreen algae” refers to multiple cells of a single Aphanizomenon species,multiple cells of a single Spirulina species, or a mixture of cells frommultiple Aphanizomenon and/or Spirulina species.

“Circulatory system” as used herein refers to the mechanisms for movingblood and blood components throughout the body of a subject, includingthe vascular and lymph systems. The mechanisms of the circulatory systeminclude, but are not limited to, the heart, blood vessels (arteries,veins, and capillaries), and lymph vessels.

“Colostrum” as used herein refers to a fluid secreted by the mammaryglands of female mammals during the first few days of lactation,containing various nutrients and protease inhibitors that keep it frombeing destroyed by the processes of digestion. Humans produce relativelysmall amounts of colostrum in the first two days after giving birth, butcows produce about nine gallons of colostrum. Colostrum containsconcentrated levels of important immune modulators, including TransferFactor, PRP, IGF-1, n-acetyl neuraminic acid, GMP, nucleic acid anddefensins. Colostrum extracts have been shown to activate phagocytosisby monocytes and increase the reactive oxygen burst in polymorphnucleated cells. Colostrum was also shown to trigger natural killer (NK)cell activation and also trigger the secretion of anti-inflammatorycytokines in in vitro cell-based assays. References herein to colostrumalso include derivatives and artificial substitutes thereof.

“Component of Polygonum multiflorum” as used herein refers to anyfraction, extract, or isolated or purified molecule from Polygonummultiflorum. For example, the component is a protein or nucleic acid ora polysaccharide, a phytochemical, or a fraction of Polygonummultiflorum. Thus, in certain embodiments of the invention, componentsof Polygonum multiflorum are obtained by disrupting Polygonummultiflorum, adding an inorganic or organic solvent, and collectingfractions. Specific, non-limiting examples of fractions are isolatedusing high performance liquid chromatography, thin layer chromatography,or distillation. Fractionation may be based on the molecular weight orthe hydrophobicity of the components of Polygonum multiflorum. Examplesof components found in Polygonum multiflorum include hydroxyl stilbenes,anthraquinones and derivatives, lecithin, chrysophanic acid, emodin,rhein, chrysophanic acid anthrone, and2,3,5,4′-tetrahydroxystilbene-2-O-βD-glucoside, among others.

“Component of Lycium barbarum” as used herein refers to any fraction,extract, or isolated or purified molecule from Lycium barbarum. Forexample, the component is a protein or nucleic acid or a polysaccharide,a phytochemical, or a fraction of Lycium barbarum. Thus, in certainembodiments of the invention, components of Lycium barbarum are obtainedby disrupting Lycium barbarum, adding an inorganic or organic solvent,and collecting fractions. Specific, non-limiting examples of fractionsare isolated using high performance liquid chromatography, thin layerchromatography, or distillation. Fractionation may be based on themolecular weight or the hydrophobicity of the components of Lyciumbarbarum.

“Differentiation” as used herein refers to the process by which cellsbecome more specialized to perform biological functions. For example,hematopoietic stem cells, hematopoietic progenitors and/or stem cellsmay change from multipotent stem cells into cells committed to aspecific lineage and/or cells having characteristic functions, such asmature somatic cells. Differentiation is a property that is oftentotally or partially lost by cells that have undergone malignanttransformation.

“Enhancement,” “enhance” or “enhancing” as used herein refers to animprovement in the performance of or other physiologically beneficialincrease in a particular parameter of a cell or organism. At times,enhancement of a phenomenon is quantified as a decrease in themeasurements of a specific parameter. For example, migration of stemcells may be measured as a reduction in the number of stem cellscirculating in the circulatory system, but this nonetheless mayrepresent an enhancement in the migration of these cells to areas of thebody where they may perform or facilitate a beneficial physiologicresult, including, but not limited to, differentiating into cells thatreplace or correct lost or damaged function. In one embodiment,enhancement refers to a 15%, 20%, 30% or greater than 50% reduction inthe number of circulating stem cells. In one specific, non-limitingexample, enhancement of stem cell migration may result in or be measuredby a decrease in a population of the cells of a non-hematopoieticlineage, such as a 15%, 20%, 30%, 50%, 75% or greater decrease in thepopulation of cells or the response of the population of cells. In oneembodiment, an enhanced parameter is the trafficking of stem cells. Inone embodiment, the enhanced parameter is the release of stem cells froma tissue of origin. In one embodiment, an enhanced parameter is themigration of stem cells. In another embodiment, the parameter is thedifferentiation of stem cells. In yet another embodiment, the parameteris the homing of stem cells.

“Fucoidan” as used herein describes sulfated fucans obtained from algae.Fucoidan has been obtained from a broad range Algae species as providedin the following non-exhaustive list: Cladosiphon okamuranus, Chordariaflagelliformis, Ch. Gracilis, Saundersella simplex, Desmaestiaintermedia, Dictyosiphon foeniculaceus, Dictyota dichotoma, Padinapavonica, Spatoglussum, schroederi, Adernocystis utricularis, Pylayellalittoralis, Ascophyllum nodosum, Bifurcaria bifurcata, Fucus.Visculosus, F. spiralis, F. serratus, F. evaescens, Himanthalia lorea,Hizikia fusiforme, Pelvetia canaliculata, P. wrightii, Sargassumstenophyllum, S. honeri, S. Khellmanium, S. muticum, Alaria fistulosa,A. marginata, Arthrothammus bifidus, Chorda film, Ecklonia kurome, E.cava, Eisenia bicyclis, Laminaria angustata, L. brasiliensis, L.cloustoni, L. digitata, L. japonica, L. religiosia, L. saccharina,Macrocystis integrifolia, M. pyrifera, Nereocystis luetkeana, Undariapinnatifida, Petalonia fascia, Scytosiphon lomentaria. Substantialpharmaceutical research has been done on fucoidan, focusing primarily ontwo distinct forms: F-fucoidan, which is >95% composed of sulfatedesters of fucose, and U-fucoidan, which is approximately 20% glucuronicacid, each of which is included in the term “fucoidan” as used herein.Depending on the source of the fucoidan, fucoidan can serve as areleasing agent in certain embodiments, while in other embodiments,fucoidan can serve as a migration agent.

“Hematopoiesis” as used herein refers to the formation and developmentof blood cells. Prenatally, hematopoiesis occurs in the yolk sack, thenliver, and eventually the bone marrow. In normal adults, it occursprimarily in bone marrow and lymphatic tissues. All blood cells developfrom pluripotent stem cells, which are committed to three, two, or onehematopoietic differentiation pathways. This includes the production ofhematopoietic cells including B-cells, T-cells, cells of the monocytemacrophage lineage, and red blood cells.

“Hematopoietic agent” as used herein refers to a compound, antibody,nucleic acid molecule, protein, cell or other molecule that affectshematopoiesis. A molecular agent can be a naturally-occurring moleculeor a synthetic molecule. In some instances, the agent affects thegrowth, proliferation, maturation, migration or differentiation orrelease of hematopoietic cells. In various embodiments, the agent isPolygonum multiflorum or an extract or component of Polygonummultiflorum.

“Hematopoietic stem cells” as used in the present invention meansmultipotent stem cells that are capable of eventually differentiatinginto all blood cells including, erythrocytes, leukocytes,megakaryocytes, and platelets. This may involve an intermediate stage ofdifferentiation into progenitor cells or blast cells. The term“hematopoietic progenitors”, “progenitor cells” or “blast cells” areused interchangeably in the present invention and describe maturing HSCswith reduced differentiation potential, but are still capable ofmaturing into different cells of a specific lineage, such as myeloid orlymphoid lineage. “Hematopoietic progenitors” include erythroid burstforming units, granulocyte, erythroid, macrophage, megakaryocyte colonyforming units, granulocyte, erythroid, macrophage, and granulocytemacrophage colony-forming units.

“Homing” as used herein refers to the process of a cell migrating fromthe circulatory system into a tissue or organ. In some instances, homingis accomplished via tissue-specific adhesion molecules and adhesionprocesses. Homing may refer to the migration back to the bone marrow.

“Immunologically normal” as used herein refers to a subject thatdisplays immune system characteristics typical for the species to whichthe individual belongs. These typical characteristics include, amongothers, functioning B-cells and T-cells as well as structural cellcomponents, called cell surface antigens, which act as the immunologicsignature for a particular organism.

“Immunologically compromised” as used herein refers to a subject havinga genotypic or a phenotypic immunodeficiency. Agenotypically-immunodeficient subject has a genetic defect that resultsin an inability to generate either humoral or cell-mediated responses. Aspecific, non-limiting example of a genotypically immunodeficientsubject is a genotypically immunodeficient mouse, such as a SCID mouseor a bg/nu/xid mouse. A “phenotypically-immunodeficient subject” is asubject, which is genetically capable of generating an immune response,which has been phenotypically altered such that no response is seen. Inone specific, non-limiting example, a phenotypically-immunodeficientrecipient has been irradiated. In another specific, non-limitingexample, a phenotypically-immunodeficient subject has been treated withchemotherapy. In yet another specific, non-limiting example, thephenotypically-immunodeficient subject has suffered a bacterial or viralinfection, such as the human immunodeficiency virus (HIV) or simianimmunodeficiency virus (SIV).

“Isolated biological component” (such as a nucleic acid molecule,polypeptide, polysaccharide or other biological molecule) as used hereinrefers to a biological component that has been substantially separatedor purified away from other biological components in which the componentnaturally occurs. Nucleic acids and proteins may be isolated by standardpurification methods, recombinant expression in a host cell, orchemically synthesized.

“Lycium barbarum” or “L. barbarum” as used herein refers to a smallbright orange-red, ellipsoid berry or fruit grown. One exemplary sourceis in the north of China, primarily in the Ningxia Hui AutonomousRegion. It is sometimes referred to as goji berry or wolfberry. L.barbarum belongs to the Solanaceae family, the nightshade family thatincludes hundreds of plant foods like potato, tomato, eggplant, andpeppers (paprika).

“Lymphoproliferation” as used herein refers to an increase in theproduction of lymphocytes.

“Modulation” or “modulates” or “modulating” as used herein refers toupregulation (i.e., activation or stimulation), down regulation (i.e.,inhibition or suppression) of a response or the two in combination orapart.

“Migration” as used herein refers to the central process for movement ofcells in the development and maintenance of multicellular organisms.Cells often migrate in response to, and towards, specific externalsignals, commonly referred to as chemotaxis. Migration includes theprocess of a cell moving from the circulatory system into a tissue ororgan. More specifically, circulating stem cells are tethered to thesurface of capillary endothelium via expression of adhesion molecules ofcell surfaces, resulting in cytoskeletal changes in both endothelium andstem cells, and allowing movement through the capillary wall en route toa tissue and/or organ site. In some instances, homing is accomplishedvia tissue-specific adhesion molecules and adhesion processes.

“Migration agent” as used herein are mobilization agents capable ofpromoting the process of a cell moving from the circulatory system intoa tissue or organ. Migration of stem cells may be demonstrated, forexample, by a decrease in circulating stem cells in the circulatory orimmune system, or by the expression of surface markers and/or adhesionmolecules on cell surfaces, which relate to homing, tethering, and/orextravasation of circulating stem cells to the surface of vessels suchas capillary endothelium. Examples of migration agents include isolatedor purified components extracted from Polygonum multiflorum or extractsthereof, Lycium barbarum, including a polysaccharide-rich fraction(fraction A) of Lycium barbarum extract, colostrum, including aprotein-rich fraction (fraction B) of colostrum extract, fucoidan,including an isolated component or compound extracted from an algae,such as a compound found in a polysaccharide-rich fraction (fraction C)of algae extracts, including Chordaria cladosiphon, or other algaes, orextracts thereof, mushrooms, including an isolated component or compoundextracted from a mushroom, such as a compound found in apolysaccharide-rich fraction (fraction D) of mushroom extracts,including Cordyceps sinensis or an extract thereof, Ganoderma lucidum oran extract thereof, Hericium erinaceus or an extract thereof, spirulina,including Arthrospira platensis, Arthrospira maxima, or extractsthereof. In different embodiments, this agent affects the migration ofstem cells, such as CD34^(high) (CD34+) cells. In one embodiment, themigration agent decreases the number of bone marrow-derived stem cellsand/or hematopoietic stem cells circulating in the peripheral blood. Inanother embodiment, the migration agent relates to enhanced expressionof CXCR4 on circulating stem cells.

“Mushroom polysaccharides” as used herein refers to glucans found mainlyin various species of mushrooms such as Cordyceps sinesis, Herciciumerinaceous, and Ganoderma lucidum. This also includes the numerousbioactive polysaccharides or polysaccharide-protein complexes frommedicinal mushrooms that may enhance innate and cell-mediated immuneresponses, and exhibit antitumor activities in animals and humans.

“Pharmaceutically acceptable carriers” as used herein refer toconventional pharmaceutically acceptable carriers useful in thisinvention.

“Polygonum multiflorum” or “P. multiflorum”, as used herein, refers to aspecies of herbaceous perennial vine growing to 2-4 m tall from a woodytuber native to central and southern China. Leaves are 3-7 cm long and2-5 cm broad, broad arrowhead-shaped, with an entire margin. Flowers are6-7 mm diameter, white or greenish-white, produced on short, densepanicles up to 10-20 cm long. Fruit is an achene 2.5-3 mm long. It isalso known as Fallopia multiflora, Radix Polygoni, Radix PolygoniMultiflori, fleeceflower, He Shou Wu, or Fo-Ti.

“Polysaccharide” as used herein refers to a polymer of more than aboutten monosaccharide residues linked glycosidically in branched orunbranched chains.

“Progenitor cell” as used herein refers to a cell that gives rise toprogeny in a defined cell lineage.

“Promote” and/or “promoting” as used herein refer to an augmentation ina particular behavior of a cell or organism. In one embodiment,promoting relates to the mobilization of melanocyte derived stem cells.In another embodiment, promoting relates to the differentiation of stemcells into melanocytes.

“Recruitment” of a stem cell as used herein refers to a process wherebya stem cell in the circulatory system migrates into specific site withina tissue or organ. Recruitment may be facilitated by a compound ormolecule, such as a chemoattractant signal or cell receptor. Forexample, both CXCR4 and SDF-1 have identified roles in stem cell homingand migration.

“Releasing agent” as used herein are mobilization agents capable ofpromoting the release and egress of stem cells from a tissue of origin.Release of stem cells from a tissue of origin may be demonstrated, forexample, by an increase in circulating stem cells in the circulatory orimmune system, or by the expression of markers related to egress of stemcells from a tissue of origin, such as bone marrow. Examples ofreleasing agents include fucoidan, as obtained from an extract of algaesuch as Undaria pinnatifida, and Polygonum multiflorum or extractsthereof. In one embodiment, the releasing agent increases the number ofbone marrow-derived stem cells and/or hematopoietic stem cells in theperipheral blood. In another embodiment, the releasing agent affects thenumber of stem cells, such as CD34^(high) (CD34+) cells, circulating inthe peripheral blood.

“Satellite cell” as used herein refers to a muscle-specific stem cell,often located in the periphery of muscle tissue, and capable ofmigrating into a muscle to aid in tissue repair and reconstruction.

“Stem cells” as used herein are cells that are not terminallydifferentiated and are therefore able to produce cells of other types.Characteristic of stem cells is the potential to develop into maturecells that have particular shapes and specialized functions, such asheart cells, skin cells, or nerve cells. Stem cells are divided intothree types, including totipotent, pluripotent, and multipotent.“Totipotent stem cells” can grow and differentiate into any cell in thebody and thus, can form the cells and tissues of an entire organism.“Pluripotent stem cells” are capable of self-renewal and differentiationinto more than one cell or tissue type. “Multipotent stem cells” areclonal cells that are capable of self-renewal, as well asdifferentiation into adult cell or tissue types. Multipotent stem celldifferentiation may involve an intermediate stage of differentiationinto progenitor cells or blast cells of reduced differentiationpotential, but are still capable of maturing into different cells of aspecific lineage. The term “stem cells”, as used herein, refers topluripotent stem cells and multipotent stem cells capable ofself-renewal and differentiation. “Bone marrow-derived stem cells” arethe most primitive stem cells found in the bone marrow which canreconstitute the hematopoietic system, possess endothelial, mesenchymal,and pluripotent capabilities. Stem cells may reside in the bone marrow,either as an adherent stromal cell type, or as a more differentiatedcell that expresses CD34, either on the cell surface or in a mannerwhere the cell is negative for cell surface CD34. “Adult stem cells” area population of stem cells found in adult organisms with some potentialfor self-renewal and are capable of differentiation into multiple celltypes. Other examples of stem cells are marrow stromal cells (MSCs),HSC, multipotent adult progenitor cells (MAPCs), very smallembryonic-like stem cells (VSEL), epiblast-like stem cell (ELSC) orblastomere-like stem cell (BLSC).

“Stem cell circulation agent” (SCCA), “mobilization agent”, and/or“mobilization factor” as used herein refers to one or more compounds,antibodies, nucleic acid molecules, proteins, polysaccharides, cells, orother molecules, including, but not limited to, neuropeptides and othersignaling molecules, that affects the release, circulation, homingand/or migration of stem cells from the circulatory system into tissueor organ. A molecular agent may be a naturally occurring molecule or asynthetic molecule. Examples of mobilization agents include “releasingagents”, wherein a releasing agent is capable of promoting the egress ofstem cells from a tissue of origin and also “migration agents”, whereina migration agent is capable of promoting the process of a cell movingfrom the circulatory system into a tissue or organ.

“Subject” as used herein includes all animals, including mammals andother animals, including, but not limited to, companion animals, farmanimals and zoo animals. The term “animal” can include any livingmulti-cellular vertebrate organisms, a category that includes, forexample, a mammal, a bird, a simian, a dog, a cat, a horse, a cow, arodent, and the like. Likewise, the term “mammal” includes both humanand non-human mammals.

“Therapeutically effective amount” as used herein refers to the quantityof a specified composition, or active agent in the composition,sufficient to achieve a desired effect in a subject being treated. Forexample, this can be the amount effective for enhancing migration ofstem cells that replenish, repair, or rejuvenate tissue. In anotherembodiment, a “therapeutically effective amount” is an amount effectivefor enhancing trafficking of stem cells, such as increasing release ofstem cells, as can be demonstrated by elevated levels of circulatingstem cells in the bloodstream. In still another embodiment, the“therapeutically effective amount” is an amount effective for enhancinghoming and migration of stem cells from the circulatory system tovarious tissues or organs, as can be demonstrated be decreased level ofcirculating stem cells in the bloodstream and/or expression of surfacemarkers related to homing and migration. A therapeutically effectiveamount may vary depending upon a variety of factors, including but notlimited to the physiological condition of the subject (including age,sex, disease type and stage, general physical condition, responsivenessto a given dosage, desired clinical effect) and the route ofadministration. One skilled in the clinical and pharmacological artswill be able to determine a therapeutically effective amount throughroutine experimentation.

“Trafficking” as used herein refers to the process of movement of a cellfrom the tissue of origin, traveling within the circulatory or immunesystem, and localization towards a site within a tissue and/or organ.Trafficking also includes stem cell mobilization, beginning with releasefrom a tissue of origin, such as egress of stem cells from bone marrow.Trafficking further includes movement of a cell from the tissue oforigin, homing by adhesion to the endothelium, transmigration, and finalmigration within the target tissue and/or organ. Furthermore,trafficking may include the process of movement of a cell of the immunesystem. One specific, non-limiting example of trafficking is themovement of a stem cell to a target organ, also referred to asmigration. Another specific, non-limiting example of trafficking is themovement of a B-cell or a pre-B-cell leaving the bone marrow and movingto a target organ.

“Treat,” “treating” and “treatment” as used herein refer to boththerapeutic treatment and prophylactic or preventative measures, whereinthe object is to prevent or slow down (lessen) the targeted condition,disease or disorder (collectively “ailment”) even if the treatment isultimately unsuccessful. Those in need of treatment may include thosealready with the ailment as well as those prone to have the ailment orthose in whom the ailment is to be prevented.

As described, stem cells are unique cells that possess the capacity todifferentiate into more specialized cells. One particular type of stemcell, hematopoietic stem cells (HSCs), are capable of differentiatinginto many different types of blood cells. In addition, HSCs typicallyreside in the bone marrow, where proliferation and self-renewal of thecells allows HSCs to be involved in the support and maintenance of thehematopoietic system. Existing scientific literature has chiefly focusedon HSCs' potential to develop into hematopoietic lineage cellsderivatives. Emerging evidence has further identified the capacity forHSCs to also differentiate into non-hematopoietic, tissue specificcells. Recently, HSCs have been found to possess the capacity todifferentiate into a variety of tissue-specific cell types, such asmyocytes, hepatocytes, osteocytes, glial cells, and neurons. As aresult, HSCs form blood and immune cells that are responsible forconstant maintenance and immune protection of virtually every cell typeof the body.

Similarly, bone marrow stem cells (BMSCs) were recently shown to havesignificant capability to become cells of other tissues. In the bonemarrow, stem cells duplicate using a process known as “asymmetricalcellular division” according to which the two daughter cells are notidentical; one cell retains the original DNA and remains in the bonemarrow whereas the other cell contains the DNA copies and is released inthe blood where it migrates into various tissues in need of repair.BMSCs have been traditionally considered to have little potential forplasticity, being limited in their development to red blood cells,lymphocytes, platelets, bone and connective tissue. However, muchscientific work has been published over the past few years thatdemonstrates the exceptional plasticity of BMSC. For example, aftertransplantation, BMSCs and HSCs were shown to have the ability to becomemuscle cells (Abedi et al., 2004), heart cells (Fraser et al., 2004),endothelium capillary cells (Asahara et al., 1999), liver cells (Jang etal., 2004), as well as lung, (Krause et al., 2001), gut (Krause et al.,2001), skin (Branski et al., 2008), and brain cells (Dezawa et al.,2005). As a further illustrative example, Jang et al. (2004) performedan experiment which not only demonstrated the ability of HSC to becomeliver cells upon contact with specific liver-derived molecules, but thisprocess took place within hours. Briefly, HSCs were co-cultured witheither normal or damaged liver tissue separated by a semi-permeablemembrane (pores large enough to let molecules pass through, but smallenough to prevent the passage of cells from one compartment to theother, pore size 0.4 μm). Using immunofluorescence assay methods todetect molecules specific for either HSCs (CD45) or liver cells(albumin), the researchers could follow the transformation of thepopulation of cells placed in the upper compartment. When HSCs werecultured alone for 8 hours, they only expressed CD45 and no albumin,indicating that no HSCs had differentiated into liver cells. However,when HSCs were exposed to injured liver tissue, they rapidly becamepositive for albumin. Over time, the population of cells positive forCD45 began to decrease as the population positive for albumin began toincrease. Albumin-positive cells were seen as early as 8 hours into theprocedure and increased in frequency to 3.0% at 48 hours. The conversionwas minimal and delayed when HSCs were exposed to undamaged liver(control for injury).

Because HSCs and BMSCs play an important role in the healing andregenerative processes of various tissues and organs in the body beyondtheir traditional role in maintaining hematopoietic and immune systemsof the body, activation and enhancement of stem cell trafficking mayamplify these physiological processes and provide a potential therapyfor various pathologies. The classic source of HSCs and BMSCs is bonemarrow, which includes hip, ribs, sternum and other bone structures.Bone provides a unique regulatory microenvironment for HSCs and BMSCs,including interaction with a specific mesenchymal cell type (theosteoblast), extracellular matrix glycoproteins and a uniquely richmineral signature. (Adams and Scadden, 2006) This stem cell “niche”contains a great deal of critical molecular interactions which guide theresponse of stem cells to specific physiological conditions. The nichemay be an important focal point for changes in the state of tissue thatresult in a change in the regenerative processes rooted in stem cellactivity. (Adams and Scadden, 2006)

Beyond populations of HSCs found in bone marrow, HSCs are also presentin the peripheral bloodstream of normal, healthy persons. It has beenknown for decades that a small number of stem and progenitor cellscirculate in the bloodstream, but more recent studies have shown thatgreater numbers of HSCs can be coaxed into mobilization from marrow toblood by injecting the donor with a cytokine, such as granulocyte-colonystimulating factor (G-CSF). Despite this advance, the natural process bywhich stem cells are released from bone marrow and migrate towards asite within tissue and/or an organ is not fully understood. A leadingmodel involves the chemokine, Stromal-Derived Factor-1 (SDF-1) and itsspecific receptor, CXCR4. In this capacity, the binding of SDF-1 toCXCR4, leads to adherence of stem cells to bone marrow through increasedexpression of adhesion molecules on the cell membrane surface.Disruption of adhesion of stem cells to bone marrow thus promotesmobilization of stem cells into the peripheral bloodstream. (FIG. 1C)Some factors such as G-CSF or IL-8 may interfere with adhesion throughelevated activation of protelytic enzymes or degradation of the SDF-1ligand. (Drapeau 2010) Other types of molecules, such as L-selectinblockers, may instead down-regulate CXCR4 expression which in turnreduces stem cell adhesion to the bone marrow environment. Generallyspeaking enhancing binding of SDF-1 to CXCR4 promote adherence,therefore L-selectin blockers such as sulfated fucans, which reducesCXCR4 expression, can trigger stem cell mobilization. (Drapeau 2010)

Stem cells circulating in the peripheral bloodstream are recruited tosites of tissue in need of repair and regeneration through homing andextravasation. This mobilization of stem cells into the bloodstream andsubsequent migration to the site of tissue injury results from acombination of mechanical and chemoattractant signals. (Drapeau 2010)Mechanical force or other factors may activate L-selectins on thesurface of stem cells. Activation of L-selectins, in turn, may promoteelevated expression of the receptor, CXCR4. Cells at the site of tissueinjury may also secrete SDF-1 ligand, thereby attracting stem cellsexpressing receptor CXCR4 to the injury site. The interaction of SDF-1and CXCR4 promotes sufficient adhesion to halt circulation of a stemcell in the peripheral blood stream. (FIG. 1B) Based on this model,L-selectin blockers such as sulfated fucans, may possess a criticalcapacity to mobilize HSCs into the bloodstream, with subsequent homing,extravasation and migration into tissue promoting regenerativemaintenance and repair of cells and tissues in an organism. WhereasG-CSF is released from injured tissue and its presence in thebloodstream triggers HSC release from bone marrow, dietary supplementscomposed of L-selectin blockers may possibly support the phenomenon ofnatural regeneration and repair in the body.

Remarkably, in ancient traditional Chinese medicine (TCM) literature,there is description of similar processes, including a concept calledthe Jing. Although the term has changed over time and today refers toessentially to “semen”, ancient texts referred to two different Jing:one primordial that was present at the time of conception and led to theformation of the fetus, and the other that remained in the body and wasregenerative and healing. Modernly, the notion of an innate healing andregenerative capacity, as exemplified by the TCM concept of Jing, isconsistent with the remarkable capacity of embryonic and adult stemcells to facilitate these processes. (Yang et al. 2009) It isinteresting to note that herbs known to support the Jing are used to“tonify the blood”, turn back hair to their natural color, and foroverall regenerative properties. An example is goji berry (Lyciumbarbarum), which is known to support the Jing and is used for longevity;goji berry has been shown to support stem cell migration. Anotherexample is StemEnhance, a supplement containing blue-algae Aphanizomenonflos aquae (AFA), that is known to support stem cell mobilization, hasbeen was shown to help reverse hair color and support overall healingand regeneration. (Jensen et al., 2007) As compositions such asStemEnhance influence stem cell activity in the body, these effects maybe understood as stimulating the Jing. Conversely, it is of greatinterest to understand if TCM medicines and herbs known to stimulate theJing can be demonstrated as possessing effects on stem cell activitiessuch as proliferation, migration and mobilization. The inventorstherefore studied the effects of a widely used herb to stimulate theJing, Polygonum multiflorum, with a particular view towards effects onstem cell migration and mobilization.

Polygonum multiflorum.

The dried root tuber of Polygonum multiflorum plant, also known asfleeceflower root, has been used as a traditional Chinese medicinecalled He shou wu, this medication gaining notoriety in TCM from a taleof a famous Chinese military officer condemned to death and jailedwithout food or drink. Surviving by consuming the leaves and roots ofthe vinelike weed, Polygonum multiflorum, the officer's captors laterfound his remains as still having lustrous black hair. While the originsof this tale are apocryphal, they serve to illustrate the long-heldnotion that Polygonum multiflorum possesses important properties fortapping into the regenerative and restorative potential of the body.Recent scientific studies have confirmed that extracts of Polygonummultiflorum are indeed capable of promoting hair follicle growth,through increased expression of sonic hedgehog (Shh) and β-cateninexpression—two important pathways involved in both early embryogenesisand maintaining stem cell identity. (Park et al. 2011)

Further analysis of Polygonum multiflorum extracts have confirmed thisplant to be a rich source of bioactive compounds, two notable examplesbeing anthraquinones and derivatives and hydroxy stilbenes.Anthraquinones and derivatives have served as the basis forantimalarial, laxative, and chemotherapy treatments. Hydroxyl stilbenes,such as 2,3,5,4′-tetrahydroxystilbene-2-O-β-D-glucoside, have been showto provide important neuroprotective effects warding off symptoms ofdifferent neurodegenerative diseases. Together, these results indicatethat components of Polygonum multiflorum extracts possess importantproperties for healing and regenerating the body, possibly by modulatinginflammation, reducing risk of cancer proliferation, and/or providingprotective effects for cells, tissues, and organs of the body.

While effects of these components in Polygonum multiflorum is somewhatunderstood for certain specific conditions, there is much less knowledgeabout how components of Polygonum multiflorum may specifically influencestem cell activity in the body. This is surprising given that, asdescribed, stem cells play an integral role in the body's naturalhealing and regeneration mechanisms. One of the few existing studies onthe subject indicates that Polygonum multiflorum extracts promotesproliferation of stem cells and progenitors, as shown by an increase inthe number of bone marrow stem cells and lymphoid progenitors followingadministration of Polygonum multiflorum extracts in mice. (Zhiweng etal. 1991). Similarly, U.S. patent application Ser. No. 12/006,221describes an increase in GM-CSF and stem cell factor (SCF) expressionfollowing administration in mice. These results present intriguingquestions about potential effects of Polygonum multiflorum extracts onstem cell activity, given that both GM-CSF and SCF are implicated asplaying important roles in stem cell migration and mobilization, asdescribed above.

Fucoidan.

Fucoidan is a sulfated fucan polysaccharide L-selectin agonist that wasdocumented to promote the egress of HSCs from compartments in bonemarrow into the peripheral blood stream upon intravenous injection,although this effect seemed unrelated to its stimulation of L-selectin(Frenette et al., 2000). Circulation of HSCs in the peripheralbloodstream is a critical step in promoting the stem cell regenerationand repair mechanisms in the body. As a sulfated fucan, fucoidan isfound in various species of algae. Other sulfated fucans have also beenfound in animal species, such as echinoderms (e.g., sea urchins and seacucumbers).

Despite in vivo data in animal models that demonstrate significantlyelevated levels of HSCs following intravenous fucoidan administration,observations of positive clinical effects in human subjects are muchmore limited. Reported studies have shown that the percentage of HSCsexpressing an important trafficking receptor, CXCR4, increasedsignificantly following 4 days of oral fucoidan administration. (Irhimehet al., 2007) However, only a slight change was observed in the absolutenumber of HSCs circulating in peripheral blood.

As described, fucoidan (also known as fucoidin or fucansulfate in theart) is a sulfated fucose polysaccharide L-selectin ligand. Selectinactivity depends on important carbohydrate or polypeptide modificationssuch as sialylation, fucosylation, and sulfation. The presence ofbinding sites for sulfated fucans such as fucoidan on P- and L-Selectinhas been demonstrated to be at least partially the mechanism by whichfucoidan promotes detachment of HSCs from BM. (Frenette et al., 2000,2461, Jensen et al., 2007, 190) Perhaps more significantly, sulfatedfucans such as fucoidan, have been shown to displace SDF-1 sequesteredon endothelial surfaces or bone marrow through completive binding to aheparin-binding domain present on SDF-1. Occupation of theheparin-binding site of SDF-1 by fucoidan prevents tethering to cellsurfaces, thereby increasing circulating SDF-1 levels in plasma.(Sweeney et al., 2008) Without being bound by any particular theory, theenhanced levels of SDF-1 ligand in the bloodstream may thus promoteegress of CXCR4 receptor expressing HSCs from the BM. (Sweeney et al.,2008) (FIG. 1C) Based on this model, the inventors hypothesized thatL-selectin ligand, such as fucoidan, may possess a critical capacity tomobilize HSCs and oral administration of dietary supplements composed offucoidan may best support natural regeneration and repair in the body.

Compelling in vivo data in animal models demonstrates significantlyelevated levels of circulating HSCs following intravenous (IV) fucoidanadministration in mice and primates, although significant drawbackswould present limitations for human therapeutic use. Recent reports haveshown a dramatic 12-fold increase in levels of circulating HSCs, HSCprogenitors and derivative cell types (including erythroid burst formingunits, granulocyte, erythroid, macrophage, megakaryocyte colony formingunits, granulocyte, erythroid, macrophage, and granulocyte macrophagecolony-forming units) compared to untreated controls, 3 hours followinginjection of fucoidan (source unknown) into mice. Similar results ofsustained elevation in levels of HSCs, HSCs progenitors and derivativecell types, were reported after daily injections for 3 days. (Sweeney etal., 2008) Injection of fucoidan in primates has also been demonstratedto increase HSCs and HSC-derivative levels by 11-26 fold after 6 hoursafter administration, with sustained elevation still observable up to 24hours later. (Sweeney et al., 2000) Despite these positive observations,several challenges could impede therapeutic use of fucoidan in humansubjects. The temporary and transitory effect of elevated HSCscirculating and bone marrow-derived stem cells may fail to fully realizethe positive clinical benefits of stem cell regenerative and repairmechanisms, since sustained or repeated periods of elevation may beneeded to enable stem cell homing and extravasation processes thatunderlie therapeutic stem cell activity. This limitation is furthercompounded in view of the difficulty and inconvenience of routinelyadministering IV injections.

Existing observations in human subjects are limited and available dataon oral fucoidan administration in humans does not mirror the positiveclinical effects of animal studies using IV injection. Reported studiesby others have shown that the percentage of HSCs expressing an importanttrafficking receptor, CXCR4, increased significantly (45% to 90%) after12 days of oral fucoidan administration (3 grams daily of 10% w/w or 75%w/w fucoidan extracts from Undaria pinnatifida). However, only a slightchange (˜12%) was observed in the absolute number of HSCs circulating inperipheral blood (maximal effect was 1.64 to 1.85 cells/pi after 4 daysof fucoidan extract administration). (Irhimeh et al., 2007) Importantly,for therapeutic applications involving oral administration, fucoidan iscapable of surviving acidic conditions in the stomach and does notdemonstrate adverse side effects. (Irhimeh et al., 2007) This isconsistent with reports that catalytic fucoidinase, which metabolizesfucoidan, is found only in marine interverbrates and not terrestrialmammals. (Berteau and Mulloy, 2003) This may provide an vitaltherapeutic benefit of high persistence and stability of an administeredsulfated fucan, including fucoidan, for sustained therapeutic effect. Itis particularly ideal for oral uses where diffusion into the bloodstreammust first survive enzymatic processing in the mouth, esophagus, andintestines, in addition to the highly acidic conditions of the stomach.

The inventors have discovered that the source of fucoidan andappropriate dosing regimens are critical features for promoting HSCmobilization through oral fucoidan administration. Fucoidan is a memberof the broader class of sulfated fucans, which are polysaccharides richin L-fucose and obtained primarily from two sources: algae and marineinvertebrates. Sulfated fucans obtained from these two sources differgreatly in composition and structure. This diversity of molecularstructure further exists across fucoidans from different species ofalgae. While generally described as ˜20,000 molecular weightpolysaccharide composed of L-fucose, exact fucoidan structures dependin-part, on the source organism. As example, the most well-studiedfucoidan from F. vesculosus, is reported to be composed primarily ofL-fucose with α(1→3) glycosidic bonds and sulfate groups at position 4,with sulfated fucose branches every 5 units. In contrast, fucoidan froma different algae, Ascophylum nodosum, has a large proportion ofrepeating α(1→3) and α(1→4) glycosidic bonds that alternate foroligosaccharide formation, possibly with few sulfated branching pointsas showing in nuclear magnetic resonance (NMR) studies (Berteau, 2003).In sum, fucoidans from different species are structurally distinct,heterogeneous and diverse.

The present invention provides new compositions and methods forproviding a wide range of clinical and physiological benefits to asubject in need thereof by the administration of a mobilization agent.While not wishing to be bound by any particular theory, the inventorsbelieve that the beneficial and other physiological results obtainedthrough administration of the inventive compositions result fromenhancing stem cell trafficking and migration that follows theadministration of the mobilization agent.

In various embodiments, the mobilization agent comprises one or morecomponents selected from the group including: Polygonum multiflorum,Lycium Barabrum, blue-green algae (e.g. Aphanizomenon flos aquae (AFA)),colostrum, mushroom polysaccharides (e.g., Cordyceps sinensis, Hericiumerinaceus (Lion's mane), Ganoderma lucidum (Reishi)), fucoidan(optionally extracted from algaes, e.g., Undaria pinnatifida, Chordariacladosiphon (Limu)), spirulina (e.g., Arthrospira platensis, Arthrospiramaxima), analogs thereof, derivatives thereof, extracts thereof,synthetic or pharmaceutical equivalents thereof, fractions thereof, andcombinations of any of the foregoing items. The mobilization agents maybe combined together in one or more compositions or they may beadministered or consumed separately as part of a regimen. They may haveindividual physiological effects, additive effects and/or synergisticeffects with one another, such as serving as both a releasing agent andmigration agent. In some embodiments, the mobilization agent is capableof functioning as a migration agent, promoting the process of a cellmoving from the circulatory system into a tissue or organ. In someembodiments, the mobilization agent is capable of functioning as areleasing agent, promoting the release and egress of stem cells from atissue of origin.

In one embodiment, a mobilization agent is administered to a subject,for example Polygonum multiflorum, though the subject may be provided amixture of Polygonum multiflorum, and other mobilization agents. In someembodiments, the subject consumes and digests whole Polygonummultiflorum root, leaves, stem, seeds, fruits, and/or other plant parts.The whole Polygonum multiflorum root, leaves, stem, seeds, fruits,and/or other plant parts may be fresh, frozen, freeze-dried, dehydrated,fermented, or preserved in some other manner. Therefore, Polygonummultiflorum, as described herein, encompasses whole Polygonummultiflorum root, leaves, stem, seeds, fruits, and/or other plant parts.In other embodiments, the mobilization agent is an extract of Polygonummultiflorum, or an isolated component or compound extracted fromPolygonum multiflorum, such as a compound found in a polysaccharide-richfraction of Polygonum multiflorum extracts, or a fraction soluble inaqueous solutions, or a fraction soluble in organic solvents. Polygonummultiflorum can be provided alone as an isolated or purified substance,or may be part of a composition including a pharmaceutically acceptablecarrier. In one embodiment, Polygonum multiflorum or extracts thereof iscapable of functioning as a migration agent. In one embodiment,Polygonum multiflorum or extracts thereof is capable of functioning as areleasing agent.

Extracts of components found in Polygonum multiflorum includeanthraquinones and derivatives, hydroxyl siltbenes, lecithin,chrysophanol, chrysophanic acid, chrysophanol anthrone, emodin,physcion, rhein, chrysophanic acid anthrone, resveratrol, piceid,2,3,5,4′-tetrahydroxystilbene-2-O—O-D-glucopyranoside,2,3,5,4′-tetrahydroxystilbene-2-O-β-D-glucopyranoside-2″-O-mo-nogalloylester,2,3,5,4′-tetrahydroxystilbene-2-O-β-D-glucopyranoside-3″-O-monogalloylester, 2,3,5,4′-tetrahydroxystilbene-2-O-β-D-glucoside, gallic acid,catechin, epicatechin, 3-O-galloyl(−)-catechin,3-O-galloyl(−)-epicatechin, 3-O-galloyl-procyanidinB-2,3,3′-di-O-galloyl-procyanidin B-2, and β-sitosterol.

The identity and nature (e.g., stability) of components in preparedPolygonum multiflorum extracts may vary depending on the method used forextraction. For example, water extraction is a leading method ofexacting components from Polygonum multiflorum. However, certaincomponents, such as anthraquinones and derivatives are very insoluble inwater. Anthraquinones and derivatives are also insoluble in organicsolvents at room temperature, but soluble in hot organic solvents (e.g.,boiling temperature), such as methanol or ethanol. Similarly,2,3,5,4′-tetrahydroxystilbene-2-O-β-d-glycoside is known to readilydegrade in aqueous solutions in a temperature and pH dependent manner.(Ren et al. 2011) Therefore, it is understood that extracts of Polygonummultiflorum may be prepared according to any method known in the art.This includes, water extraction, organic solvent extraction (e.g., U.S.patent application Ser. No. 12/006,221), or combinations of suchexemplary methods (e.g., admixtures). Examples of organic solvents thatmay be used include methanol, n-hexane, ethyl acetate, and n-butanol.Combinations of two or more water and/or organic solvents could be addedtogether to generate additional partition layers for extractingdifferent components in different partition layers. Similarly, extractsfrom Polygonum multiflorum may be prepared from fresh, unprocessed wholeplants or parts thereof, or extracts may be prepared from processedPolygonum multiflorum whole plants or parts thereof. For example,processing may be performed by any known method in the art, one examplebeing fermentation. Processing may improve bioavailability of thecomponents in extracts from Polygonum multiflorum, such as throughfermentation with bacteria such as Lactobacillus sp. (Park et al., 2011)or through addition of black beans.

In one embodiment, a mobilization agent is administered to a subject,for example a blue-green algae such as Aphanizomenon flos aquae (AFA),though the subject may be provided a mixture of blue-green algae andother mobilization agents. In some embodiments, the subject consumes anddigests whole blue-green algae. Blue-green algae may be fresh, frozen,freeze-dried, dehydrated, or preserved in some other manner. In oneembodiment, the mobilization agent is an extract of blue-green algae, oran isolated component or compound extracted from blue-green algae, suchas a compound found in a polysaccharide-rich fraction of blue-greenalgae extract, or a compound in a water soluble compartment of anblue-green algae extract. Blue-green algae can be provided alone as anisolated or purified substance, or may be part of a compositionincluding a pharmaceutically acceptable carrier. In one embodiment,blue-green algae is capable of functioning as a migration agent. In oneembodiment, blue-green algae is capable of functioning as a releasingagent.

In one embodiment, a mobilization agent is administered to a subject,for example Lycium barbarum, though the subject may be provided amixture of Lycium barbarum and other mobilization agents. In someembodiments, the subject consumes and digests whole Lycium barbarumberries. The berries may be fresh, frozen, freeze-dried, dehydrated, orpreserved in some other manner. Therefore, Lycium barbarum, as describedherein, encompasses both whole berry, or parts of the Lycium barbarumplant. In one embodiment, the mobilization agent is an extract of Lyciumbarbarum, or an isolated component or compound extracted from Lyciumbarbarum, such as a compound found in a polysaccharide-rich fraction ofLycium barbarum extract. Lycium barbarum can be provided alone as anisolated or purified substance, or may be part of a compositionincluding a pharmaceutically acceptable carrier. In one embodiment,Lycium barbarum is capable of functioning as a migration agent. In oneembodiment, Lycium barbarum is capable of functioning as a releasingagent.

In one embodiment, colostrum is administered to a subject, though thesubject may be provided a mixture of colostrum and other mobilizationagents. In some embodiments, the subject consumes and digests wholecolostrum. The colostrum may be fresh, frozen, freeze-dried, dehydrated,or preserved in some other manner. In one embodiment, the mobilizationagent is an extract of colostrum, or an isolated component or compoundextracted from colostrum, such as a compound found in a protein-richfraction of colostrum extract colostrum can be provided alone as anisolated or purified substance, or may be part of a compositionincluding a pharmaceutically acceptable carrier. In one embodiment,colostrum is capable of functioning as a migration agent. In oneembodiment, colostrum is capable of functioning as a releasing agent.

In one embodiment, mushroom or a blend of mushrooms is administered to asubject, though the subject may be provided a mixture of mushrooms andother mobilization agents. In some embodiments, the subject consumes anddigests whole mushrooms. The mushrooms may be fresh, frozen,freeze-dried, dehydrated, or preserved in some other manner. In oneembodiment, the agent is Cordyceps sinensis or an extract thereof. Inone embodiment, the mobilization agent is Ganoderma lucidum or anextract thereof. In one embodiment, the mobilization agent is Hericiumerinaceus or an extract thereof. Mushrooms can be provided alone asisolated or purified substances, or may be part of a compositionincluding a pharmaceutically acceptable carrier. In one embodiment,mushrooms, Cordyceps sinensis, Ganoderma lucidum, and/or Hericiumerinaceus is capable of functioning as a migration agent. In oneembodiment, mushrooms, Cordyceps sinensis, Ganoderma lucidum, and/orHericium erinaceus is capable of functioning as a releasing agent.

In one embodiment, algae is administered to a subject, though thesubject may be provided a mixture of algae and other mobilizationagents. In some embodiments, the subject consumes and digests wholealgae. The algae may be fresh, frozen, freeze-dried, dehydrated, orpreserved in some other manner. In one embodiment, the mobilizationagent is Chordaria cladosiphon or an extract thereof. Algae can beprovided alone as isolated or purified substances, or may be part of acomposition including a pharmaceutically acceptable carrier. In oneembodiment, algae, Chordaria cladosiphon is capable of functioning as amigration agent. In one embodiment, algae, Chordaria cladosiphon iscapable of functioning as a releasing agent.

In one embodiment, spirulina is administered to a subject, though thesubject may be provided a mixture of spirulina and other mobilizationagents. In some embodiments, the subject consumes and digests wholespirulina. The spirulina may be fresh, frozen, freeze-dried, dehydrated,or preserved in some other manner. In one embodiment, the mobilizationagent is Arthrospira platensis, Arthrospira maxima, or an extractthereof. Spirulina can be provided alone as an isolated or purifiedsubstance, or may be part of a composition including a pharmaceuticallyacceptable carrier. In one embodiment, spirulina is capable offunctioning as a migration agent. In one embodiment, spirulina iscapable of functioning as a releasing agent.

A method is described herein for enhancing stem trafficking byadministering to a subject a therapeutically effective amount ofPolygonum multiflorum.

In one embodiment, Polygonum multiflorum, is administered to a subject,though the subject may be provided a mixture of more than oneingredient. In some embodiments, the subject consumes and digests wholeplant or parts of the plant. The Polygonum multiflorum may be fresh,frozen, freeze-dried, dehydrated, or preserved in some other manner.

In alternative embodiments, an extract of Polygonum multiflorum isprovided or administered to the subject. In another embodiment, thePolygonum multiflorum encompasses both whole plant, parts of the plant,and extracts thereof. In another embodiment, the Polygonum multiflorumcan be provided alone as an isolated or purified substance, or may bepart of a composition including a pharmaceutically acceptable carrier.In another embodiment, the extract is an anthraquinone and/orderivative. In another embodiment, the extract is a hydroxylstilbene. Inan alternative embodiment, whole Polygonum multiflorum plant isadministered to the subject. In another embodiment, parts of Polygonummultiflorum plant are administered to the subject. In one embodiment, anextract of Polygonum multiflorum is administered to the subject.

The present invention further provides a pharmaceutical preparation. Inone embodiment, the pharmaceutical preparation is 90%, 80%, 70%, 60%,50%, 40%, 30%, 20%, 10%, 5%, or 1% w/w Polygonum multiflorum or extractsthereof.

The present invention further provides a dosing regimen. In oneembodiment, the dosing regimen is dependent on the severity andresponsiveness of a disease state to be treated, with the course oftreatment lasting from a single administration to repeatedadministration over several days and/or weeks. In another embodiment,the dosing schedule is based on measurement of an active componentaccumulated in the body. In one embodiment, the active component isPolygonum multiflorum or extracts thereof. In another embodiment, thedosing regimen is dependent on the level of stem cell trafficking in thesubject. In one embodiment, the dosing regimen is dependent on theactivity of a releasing agent administered to a subject. In anotherembodiment, the dosing regimen is dependent on the number of circulatingCD34+ HSCs in the peripheral blood stream of a subject. In anotherembodiment, the dosing regimen is dependent on the number of circulatingbone marrow-derived stem cells in the peripheral blood stream of asubject. In one embodiment, the dosing regimen is 3 grams of Polygonummultiflorum or extracts thereof, administered daily. In anotherembodiment, the dosing regimen is 1 gram of Polygonum multiflorum orextracts thereof, administered daily. In another embodiment, the dosingregimen is 500 mg grams of Polygonum multiflorum or extracts thereof,administered daily. In one embodiment, the dosing regiment is 250 mggrams of Polygonum multiflorum or extracts thereof, administered daily.In another embodiment, the dosing regimen is 75 mg grams of Polygonummultiflorum or extracts thereof, administered daily. In anotherembodiment, the dosing regimen is 25 mg grams of Polygonum multiflorumor extracts thereof, administered daily.

A method is described herein for enhancing stem trafficking byadministering to a subject a therapeutically effective amount offucoidan.

In one embodiment, an algae, such as Undaria pinnatifida, isadministered to a subject, though the subject may be provided a mixtureof more than one algae. In some embodiments, the subject consumes anddigests whole algae. The algae may be fresh, frozen, freeze-dried,dehydrated, or preserved in some other manner.

In alternative embodiments, an extract of the algae is provided oradministered to the subject. In another embodiment, the algaeencompasses both whole plant and/or extracts thereof. In anotherembodiment, the algae can be provided alone as an isolated or purifiedsubstance, or may be part of a composition including a pharmaceuticallyacceptable carrier. In another embodiment, the extract is a highlysulfated, polyanionic soluble fiber. In one embodiment, the extract isan isolated fucoidan. In a different embodiment, the fucoidan ispurified following isolation. In an alternative embodiment, apolysaccharide fraction is administered to the subject. In anotherembodiment, the highly sulfated, polyanionic soluble fiber isadministered to the subject. In one, the isolated fucoidan isadministered to the subject. In a different embodiment, the purifiedfucoidan is administered to the subject. In one embodiment, Undariapinnatifida is capable of functioning as a releasing agent afteradministration to a subject.

The present invention further provides a pharmaceutical preparation. Inone embodiment, the pharmaceutical preparation is 90%, 80%, 70%, 60%,50%, 40%, 30%, 20%, 10%, 5%, or 1% w/w fucoidan.

A method is described herein for enhancing stem trafficking byadministering to a subject a therapeutically effective amount ofblue-green algae.

In one embodiment, blue-green algae, such as Aphanizomenon flos aquae(AFA) is administered to a subject, though the subject may be provided amixture of more than one algae. In some embodiments, the subjectconsumes and digests whole algae. The AFA may be fresh, frozen,freeze-dried, dehydrated, or preserved in some other manner.

In alternative embodiments, an extract of AFA is provided oradministered to the subject. In another embodiment, the AFA encompassesboth whole plant, parts of the plant, and/or extracts thereof. Inanother embodiment, the AFA can be provided alone as an isolated orpurified substance, or may be part of a composition including apharmaceutically acceptable carrier. In another embodiment, the extractis water soluble compartment. In another embodiment, the extract is apolysaccharide rich compartment.

The present invention further provides a pharmaceutical preparation. Inone embodiment, the pharmaceutical preparation is 90%, 80%, 70%, 60%,50%, 40%, 30%, 20%, 10%, 5%, or 1% w/w AFA or extracts thereof.

The present invention further provides a dosing regimen. In oneembodiment, the dosing regimen is dependent on the severity andresponsiveness of a disease state to be treated, with the course oftreatment lasting from a single administration to repeatedadministration over several days and/or weeks. In another embodiment,the dosing schedule is based on measurement of an active componentaccumulated in the body. In a certain embodiment, the active componentis fucoidan. In one embodiment, the fucoidan is isolated from Undariapinnatifida or extracts thereof. In another embodiment, the dosingregimen is dependent on the level of stem cell trafficking in thesubject. In one embodiment, the dosing regimen is dependent on theactivity of a releasing agent administered to a subject. In anotherembodiment, the dosing regimen is dependent on the number of circulatingCD34+ HSCs in the peripheral blood stream of a subject. In anotherembodiment, the dosing regimen is dependent on the number of circulatingbone marrow-derived stem cells in the peripheral blood stream of asubject. In one embodiment, the dosing regimen is 3 grams of fucoidanadministered daily. In another embodiment, the dosing regimen is 1 gramof fucoidan administered daily. In another embodiment, the dosingregimen is 500 mg grams of fucoidan administered daily. In anotherembodiment, the dosing regimen is 75 mg grams of fucoidan administereddaily. In one embodiment, the dosing regiment is 250 mg grams offucoidan administered daily.

The present invention further provides a method of enhancing thetrafficking of stem cells in a subject. In one embodiment, the level oftrafficking of stem cells relates to the number of circulating CD34+HSCs in the peripheral blood of a subject. In another embodiment, thelevel of trafficking of stem cells relates to the number of circulatingbone marrow-derived stem cells in the peripheral blood of a subject.

In another embodiment, the method provided herein enhances thetrafficking of stem cells in a subject, including administering atherapeutically effective amount of a composition containing one or moreof the following components selected from the group including: Polygonummultiflorum or extracts thereof, Lycium barbarum or extracts thereof,blue-green algae or extracts thereof, colostrum or extracts thereof,spirulina or extracts thereof, Arthrospira platensis or extractsthereof, Arthrospira maxima or extracts thereof, fucoidan, Chordariacladosiphon or extracts thereof, Hericium erinaceus or extracts thereof,Ganoderma Lucidum or extracts thereof, and/or Cordyceps Sinensis orextracts thereof, thereby enhancing the trafficking of stem cells in thesubject. In one embodiment, enhancement of stem cell trafficking may bemeasured by assaying the response of stem cells to a particular dose ofa composition containing one or more of the following componentsselected from the group including: Polygonum multiflorum or extractsthereof, Lycium barbarum or extracts thereof, blue-green algae orextracts thereof, colostrum or extracts thereof, spirulina or extractsthereof, Arthrospira platensis or extracts thereof, Arthrospira maximaor extracts thereof, fucoidan or extracts thereof, Chordaria cladosiphonor extracts thereof, Hericium erinaceus or extracts thereof, GanodermaLucidum or extracts thereof, and/or Cordyceps Sinensis or extractsthereof, thereby enhancing the trafficking of stem cells in the subject.

In another embodiment, the method provided herein enhances thetrafficking of stem cells in a subject, including administering atherapeutically effective amount of a composition containing fucoidan.In one embodiment, enhancement of stem cell trafficking may be measuredby assaying the response of stem cells to a particular dose of acomposition containing fucoidan. In one embodiment, the fucoidan is fromUndaria pinnatifida or extracts thereof.

The present invention further provides a method for enhancing thetrafficking of stem cells in a subject, comprising administering atherapeutically effective amount of a mobilization agent, therebyincreasing the release, circulation, homing and/or migration of stemcells in the subject, regardless of the route of administration.

The present invention further provides a method of inducing a transientdecrease in the population of circulating stem cells, such as CD34+ stemcells. In one embodiment, administration of a mobilization agent resultsin the migration of stem cells from the circulation to tissues fromabout 1 to about 3 hours following administration. Circulating stemcells will leave the circulatory system, thus decreasing the number ofcirculating stem cells within the subject's body. Enhancement of stemcell migration may be measured by assaying the response of stem cells toa particular dose of Polygonum multiflorum. In one embodiment, providinga mobilization agent to a subject will enhance migration of thatsubject's stem cells within a certain time period, such as less thanabout 5 hours, less than about 4 hours, less than about 2 hours, or lessthan about 1 hour following administration. In other embodiments, themobilization agent is Lycium barbarum, blue-green algae, colostrum,mushroom polysaccharides including Cordyceps sinensis, Hericiumerinaceus, Ganoderma lucidum, fucoidan including Chordaria cladosiphon,spirulina, including Arthrospira platensis, and/or Arthrospira maxima.

In another embodiment, administration of an extract of Polygonummultiflorum increases the rate of homing of stem cells measured by atransient decrease in the number of circulating stem cells within thesubject's body. In another embodiment, the percentage decrease in thenumber of circulating stem cells compared to a normal baseline may about25%, about 50%, about 75%, or even about 100% as compared to a control.In one embodiment, the control is a base line value from the samesubject. In another embodiment, the control is the number of circulatingstem cells in an untreated subject, or in a subject treated with aplacebo or a pharmacological carrier.

In another embodiment, administration of an extract of algae increasesthe rate of homing of stem cells measured by a transient decrease in thenumber of circulating stem cells within the subject's body. In anotherembodiment, the algae is Chordaria cladosiphon. In another embodiment,the percentage decrease in the number of circulating stem cells comparedto a normal baseline may about 25%, about 50%, about 75%, or even about100% as compared to a control. In one embodiment, the control is a baseline value from the same subject. In another embodiment, the control isthe number of circulating stem cells in an untreated subject, or in asubject treated with a placebo or a pharmacological carrier.

In another embodiment, administration of an extract of a mobilizationagent increases the rate of homing of stem cells measured by a transientdecrease in the number of circulating stem cells within the subject'sbody. The percentage decrease in the number of circulating stem cellscompared to a normal baseline may be about 25%, about 50%, about 75%, oreven about 100% as compared to a control. In one embodiment, the controlis a base line value from the same subject. In another embodiment, thecontrol is the number of circulating stem cells in an untreated subject,or in a subject treated with a placebo or a pharmacological carrier. Inanother embodiment, the administration of an extract of a mobilizationagent leads to an increase in CXCR4 expression on circulating stemcells.

The present invention further provides a method of inducing a transientincrease in the population of circulating stem cells, such as CD34+ stemcells following administration of Polygonum multiflorum or an extractthereof. In one embodiment, the stem cells are hematopoietic stem cells(HSGs). In another embodiment, the stem cells are bone marrow-derivedstem cells. In one embodiment, enhancement of stem cell trafficking maybe measured by assaying the response of stem cells to a particular doseof Polygonum multiflorum or an extract thereof. In one embodiment,providing Polygonum multiflorum or an extract thereof to a subject willenhance release of that subject's stem cells within a certain timeperiod, such as less than 12 days, less than 6 days, less than 3 days,less than 2, or less than 1 days. In an alternative embodiment, the timeperiod is less than 12 hours, 6 hours, less than about 4 hours, lessthan about 2 hours, or less than about 1 hour following administration.In another embodiment, the stem cells are bone marrow-derived stemcells.

In one embodiment, administration of Polygonum multiflorum or an extractthereof results in the release of stem cells into the circulation fromabout 2 to about 3 hours following administration. In anotherembodiment, released stem cells enter the circulatory system andincrease the number of circulating stem cells within the subject's body.In another embodiment, the percentage increase in the number ofcirculating stem cells compared to a normal baseline may about 25%,about 50%, about 100% or greater than about 100% increase as compared toa control. In one embodiment, the control is a base line value from thesame subject. In another embodiment, the control is the number ofcirculating stem cells in an untreated subject, or in a subject treatedwith a placebo or a pharmacological carrier.

The present invention further provides a method of inducing a transientincrease in the population of circulating stem cells, such as CD34+ stemcells following administration of an algae extract. In one embodiment,the stem cells are hematopoietic stem cells (HSGs). In anotherembodiment, the stem cells are bone marrow-derived stem cells. In oneembodiment, enhancement of stem cell trafficking may be measured byassaying the response of stem cells to a particular dose of algaeextract. In one embodiment, providing algae extract to a subject willenhance release of that subject's stem cells within a certain timeperiod, such as less than 12 days, less than 6 days, less than 3 days,less than 2, or less than 1 days. In an alternative embodiment, the timeperiod is less than 12 hours, 6 hours, less than about 4 hours, lessthan about 2 hours, or less than about 1 hour following administration.In another embodiment, the stem cells are bone marrow-derived stemcells. In one embodiment, the algae extract is from Undaria pinnatifida.

In one embodiment, administration of algae extract results in therelease of stem cells into the circulation from about 2 to about 3 hoursfollowing administration. In another embodiment, released stem cellsenter the circulatory system and increase the number of circulating stemcells within the subject's body. In another embodiment, the percentageincrease in the number of circulating stem cells compared to a normalbaseline may about 25%, about 50%, about 100% or greater than about 100%increase as compared to a control. In one embodiment, the control is abase line value from the same subject. In another embodiment, thecontrol is the number of circulating stem cells in an untreated subject,or in a subject treated with a placebo or a pharmacological carrier.

In some embodiments, the subject administered a mobilization agent ishealthy. In other embodiments, the subject is suffering from a diseaseor physiological condition, such as immunosuppression, chronic illness,traumatic injury, degenerative disease, infection, or combinationsthereof. In certain embodiments, the subject may suffer from a diseaseor condition of the skin, digestive system, nervous system, lymphsystem, cardiovascular system, endocrine system, or combinationsthereof. In specific embodiments, the subject suffers from osteoporosis,Alzheimer's disease, cardiac infarction, Parkinson's disease, traumaticbrain injury, multiple sclerosis, cirrhosis of the liver, any of thediseases and conditions described in the Examples below, or combinationsthereof. Administration of a therapeutically effective amount of amobilization agent may prevent, treat and/or lessen the severity of orotherwise provide a beneficial clinical benefit with respect to any ofthe aforementioned conditions, although the application of the inventivemethods and use of the inventive mobilization agent is not limited tothese uses. In various embodiments, the novel compositions and methodsfind therapeutic utility in the treatment of, among other things,skeletal tissues such as bone, cartilage, tendon and ligament, as wellas degenerative diseases, such as Parkinson's and diabetes. Enhancingthe release, circulation, homing and/or migration of stem cells from theblood to the tissues may lead to more efficient delivery of stem cellsto a defect site for increased repair efficiency. The novel compositionsand methods of the present invention may also be used in connection withgene therapeutic approaches.

The present invention further provides various compositions foradministration to a subject. In one embodiment, the administration istopical, including ophthalmic, vaginal, rectal, intranasal, epidermal,and transdermal. In one embodiment, the administration is oral. In oneembodiment, the composition for oral administration includes powders,granules, suspensions or solutions in water or non-aqueous media,capsule, sachets, tablets, lozenges, or effervescents. In anotherembodiment, the composition for oral administration further comprisesthickeners, flavoring agents, diluents, emulsifiers, dispersing aids orbinding agents.

Described herein are mobilization agents and methods of usingmobilization agents towards promoting stem cell trafficking. Furtherdescribed herein are migration agents and method of using migrationagents to promote the process of stem cells moving from the circulatorysystem into a tissue or organ. Also described herein are releasingagents and methods of using releasing agents to promote egress of stemcells from a tissue of origin. Also described herein is a method of oraladministration of fucoidan which results in a significant release ofHSCs into peripheral blood circulation. The inventors have demonstratedeffective administration of stem cell mobilization agents, therebyachieving a safe, convenient and effective method to enhance stemcell-related maintenance and repair in the human body. Although thepathology of stem cells is of great importance and interest, andpertains to the subject matter disclosed herein, the underlying scope ofthis invention is that the release, circulation, homing and/or migrationof stem cells from the blood to tissues is of significance in repairinginjured tissue and maintaining the vitality and health of existingtissue. Thus, the importance of developing methods and compositions forachieving this end are among the foci and aims of the present invention.

Accordingly, the present invention provides novel compositions andmethods for, among other things, enhancing natural tissue healing andrenewal in the body by supporting the trafficking of stem cells.Furthermore, the present invention provides novel compositions andmethods for preventing, slowing or otherwise diminishing the developmentof health problems in a mammal by promoting trafficking of stem cells inthe mammal. The compositions and methods disclosed herein may furtherincrease regeneration of existing tissue by supporting the release,circulation, homing and/or migration of stem cells into tissue,therefore supporting the process of tissue repair.

EXAMPLES

The following examples are provided to better illustrate the claimedinvention and are not to be interpreted as limiting the scope of thesubject matter. To the extent that specific materials are mentioned, itis merely for purposes of illustration and is not intended to limit theinvention. One skilled in the art may develop equivalent means,compositions or reactants without the exercise of inventive capacity andwithout departing from the scope of the present invention.

Example 1 Production and Preparation of L. barbarum

Polysaccharides from Lycium barbarum were prepared by the method of Luoet al. (2004). The dried fruit samples (100 g) were ground to finepowder and put in 1.5 l of boiling water and decocted for 2 h by atraditional method for Chinese medicinal herbs. The decoction was leftto cool at room temperature, filtered and then freeze-dried to obtaincrude polysaccharides.

The dried crude polysaccharides were refluxed three times to removelipids with 150 ml of chloroform:methanol solvent (2:1) (v/v). Afterfiltering the residues were air-dried. The result product was extractedthree times in 300 ml of hot water (90° C.) and then filtered. Thecombined filtrate was precipitated using 150 ml of 95% ethanol, 100%ethanol and acetone, respectively. After filtering and centrifuging, theprecipitate was collected and vacuum-dried, giving desiredpolysaccharides (13 g). The content of the polysaccharides was measuredby phenolsulfuric method (Masuko et al., 2005). Result showed that thecontent of the polysaccharides in the extract may reach 97.54%.

Example 2 Stem Cells Migrate Following Lycium barbarum Consumption

Consumption of Lycium barbarum, or compounds thereof, enhancesrecruitment and migration of CD34+ stem cells (see FIG. 2 for a diagramof stem cells entering the circulatory system).

Healthy human volunteers were identified, and the proportion of CD34+cells was evaluated in the peripheral blood (circulating CD34+ cells) ofeach person prior to consumption of Lycium barbarum and hourly for up to4 hours after consumption. The volunteers were instructed to limitphysical and mental activity for a time before and after consumption ofLycium barbarum.

Each person was provided 5 grams of dried Lycium barbarum or 1 gram ofpolysaccharide extracted from Lycium barbarum. Red blood cells in wholeblood samples obtained from each volunteer were lysed using FACS lysingsolution (Beckton Dickenson, San Jose, Calif.). The remaining cells werewashed and stained with monoclonal antibody HPCA-2 conjugated withfluorescein isothiocyanate. Samples were fixed in 1% formalin andanalyzed by flow cytometry using a FacsCalibur flow cytometer (BectonDickenson, San Jose, Calif.) and CellQuest software (Becton Dickenson,San Jose, Calif.).

FIG. 3A illustrates that consumption of Lycium barbarum triggered astrong transient decrease in circulating stem cells. Specifically, theX-axis shows the time course of a typical experiment after Lyciumbarbarum ingestion, expressed as a percentage of the control level. Atthe time of ingestion, the proportion of circulating CD34+ cells is thesame as the control. The peak decrease in circulating CD34+ cells wasobserved at about 1-2 hours after consumption. At this time point, thenumber of circulating CD34+ cells was decreased by 30% below the controlvalue. By 4 hours after Lycium barbarum ingestion, the circulating CD34+cells had returned to the baseline value. The decrease in the number ofcirculating stem cells was accompanied by an increase in the expressionof CXCR4 on the membrane of circulating stem cells (FIG. 6).

Therefore, Lycium barbarum (or a biological component of Lyciumbarbarum) can enhance the migration of endogenous stem cells (e.g. CD34+cells) from the circulation to tissues. Consumption of Lycium barbarum(or a biological component of Lycium barbarum) triggers the migrationCD34+ stem cells (e.g., see FIG. 3), thereby demonstrating the efficacyof Lycium barbarum as a migration agent.

Example 3 Stem Cells Migrate Following Colostrum Consumption

As in Example 2, and with reference to FIG. 3B, administration ofcolostrum results in stem cell migration.

Example 4 Stem Cells Migrate Following Mushroom Consumption

As in Example 2, and with reference to FIG. 4, administration of apolysaccharide rich fraction of mushroom (Cordyceps sinensis, Ganodermalucidum, Hericium erinaceus) results in stem cell migration.

Example 5 Stem Cells Migrate Following Fucoidan or Spirulina Consumption

As in Example 2, administration of fucoidan from algae seaweeds such asChordaria cladosiphon promotes certain beneficial results that mayultimately, albeit indirectly, assist with stem cell migration. Forexample, consumption of fucoidan from Chordaria cladosiphon resulted ina decrease in the number of circulating CD34+HSCs (FIG. 9), suggestingan effective role in supporting stem cell migration. As in Example 2,administration of spirulina results in stem cell migration (FIG. 4B),and administration of spirulina with Lycium barbarum, colostrum andmushrooms also results in stem cell migration (FIG. 7).

Example 6 Stem Cells Migrate Following Consumption of a Blend of LB,Colostrum, Spirulina and Mushroom

Compositions including the following components listed in Table 1 areprovided to mammalian subjects. Administration of these compositionsresults in stem cell migration.

TABLE 1 Composition 1 Composition 2 Composition 3 Composition 4 mg/dosemg/dose mg/dose mg/dose Lycium barbarum (Goji extract) 500 1,000 1,5002,000 Colostrum (Fractionated) 75 150 225 300 Spirulina 75 150 225 300Mushroom 250 500 750 1,000 Hericium erinaceus 83 166 249 332 GanodermaLucidum 83 166 249 332 Cordyceps Sinensis 83 166 249 332

Example 7 Stem Cells from Bone Marrow Populate Multiple Distant Tissues

A murine model is chosen to evaluate how a mixture of LB, colostrum andmushroom can stimulate stem cell migration into tissues, and thereforepopulate and repair distant tissues of the body.

Male mice are selected as bone marrow donor animals, while all recipientmice are females. Female recipients are sub-lethally irradiated prior toinjection of GFP+ male bone marrow cells into their tail veins. Twogroups of mice are evaluated. The first group of 20 animals aresub-lethally irradiated, injected with bone marrow, and put on normalfeed. The second group of 20 animals are also sub-lethally irradiated,receive male bone marrow, and are fed a diet of normal feed plus amixture of LB, colostrum and mushroom. Incorporation of GFP+ cells isexamined in the brain, heart muscle, muscles, liver, pancreas, sectionsof small intestine, and lung tissue

These data document the extent to which a diet containing a mixture ofLB, colostrum and mushroom promotes the homing and migration of bonemarrow stem cells to various tissues.

Example 8 Increased Stem Cell Repopulation of Traumatized Tissue

A murine model is chosen to evaluate how a mixture of LB, colostrum andmushroom can stimulate stem cell migration into tissues, and thereforepopulate and repair distant tissues of the body.

Male mice are selected as bone marrow donor animals, while all recipientmice are females. Female recipients are sub-lethally irradiated prior toinjection of GFP+ male bone marrow cells into their tail veins. Twogroups of mice are evaluated. The first group of 20 animals aresub-lethally irradiated, injected with bone marrow, and put on normalfeed. The second group of 20 animals are also sub-lethally irradiated,receive male bone marrow, and are fed a diet of normal feed plus amixture of LB, colostrum and mushroom.

After bone marrow transplant and a few days prior to the initiation ofthe feeding trial, animals are subjected to an injury such as injectionof cardiotoxin in the tibialis muscle, triggering of heart attack byligation of coronary artery, punch of the skin, laser-induced stroke, orother injuries. The recovery of mice in both groups is monitored during6 weeks using whole body fluorescence imaging. After 6 weeks, theanimals are sacrificed and the injured tissue is analyzed to assess theextent of tissue repair. Incorporation of GFP+ cells will also beexamined in the brain, heart muscle, muscles, liver, pancreas, sectionsof small intestine, and lung tissue

These data document the extent to which a diet containing a mixture ofLB, colostrum and mushroom promotes the homing and migration of bonemarrow stem cells to injured tissues, therefore enhancing the process oftissue repair and healing.

Example 9 General Study Design for Fucoidan as a Stem Cell MobilizationAgent

Two consumables were tested in human subjects: fucoidan extracted fromUndaria and a placebo. Peripheral venous blood samples were obtainedfrom healthy human volunteers between 20 and 45 years of age uponinformed consent. Blood and bone marrow samples were obtained underaseptic conditions and processed immediately. One gram of fucoidan orplacebo was given to volunteers with 4-6 oz water. Appearance of theplacebo was identical to that of the fucoidan and consisted of tan-dyed,finely ground potato flakes encapsulated in vegetable capsules.

Example 10 In Vivo Study Design

The following exclusion criteria were used: under 20 or over 65 years ofage, pregnancy, severe asthma and allergies requiring daily medication,any known chronic illness or previous/current venereal disease, frequentrecreational drug use, and impaired digestive function (includingprevious major gastrointestinal surgery). Three volunteers werescheduled on two study days one week apart. Testing was always performedat the same time of the day (8-11 a.m.) to minimize the effect ofcircadian fluctuations. Due to the interference from stress with therelease vs. homing of other types of lymphocytes, effort was taken tominimize any physical and mental stress during testing. In addition, oneach study day, volunteers were instructed to complete a questionnaireaimed at determining any exceptional stress related circumstances thatmight affect the person on that particular study day. Predeterminedcriteria for exclusion from final analysis included significant lack ofsleep and severe anxiety. After completing the questionnaire, volunteerswere instructed to remain quiescent for 4 h, comfortably seated in achair. After the first hour, the baseline blood sample was drawn.Immediately after drawing the baseline sample, a consumable wasprovided. Blood samples were later drawn 60, 90 and 180 min afteringestion of the consumable. At each time point, 5 ml of blood was drawninto heparin, and 2 ml blood was drawn into EDTA. The blood vials wereplaced on a rocking plate until use.

Example 11 Measurement of Stem Cell Populations Using FACS Sorting

The blood drawn into EDTA was used for obtaining a complete blood count(CBC) with differential, using a Coulter counter (Micro Diff II, BeckmanCoulter). All CBCs were performed within an hour of drawing the sample.All CBCs were performed in triplicate. The heparinized blood was usedfor purification of the PBMC fraction by gradient centrifugation andprocessed for immunostaining and flow cytometry. The stem cell markersCD34-FITC (clone 8G12, BD BioSciences, San Jose, Calif., USA) andCD133-PE (Miltenyi Biotech, Auburn, Calif., USA) were used for two colorimmunofluorescence. Staining of all samples with CD34-FITC/CD133-PE wasperformed in triplicate. IgG1-FITC and IgG1-PE isotype controls (BDBioSciences) were used in parallel samples. Separate, positive controlsamples for each donor included CD45-FITC and CD14-PE. Stained PBMC werefixed in 1% formalin and acquired by flow cytometry immediately. Filesof 200,000 events were collected on each triplicate sample. The percentCD34+CD133−, CD34+CD133+, and the CD34−CD133+ subsets were analyzedseparately and were analyzed again after multiplying with the lymphocytecell counts, as obtained from the average of the triplicate lymphocytecounts obtained by the CBC differential count.

Example 12 Increase in CD34+HSCs Circulating in Peripheral BloodFollowing Oral Administration of Fucoidan from Undaria pinnatifida

The inventors tested oral administration of fucoidans from severaldifferent algae species for their potential to effectuate HSCmobilization in the peripheral bloodstream of human subjects. Fucoidanfrom one species, Undaria pinnatifida, resulted in a significantelevation in the number of circulating CD34+HSCs, with increases of 17%,23% (P<0.02) and 32% ((P<0.02) occurring at 45, 90 and 180 minutemeasurement intervals, thereby demonstrating efficacy as a releasingagent. (FIG. 8) To the best of the inventors' knowledge, this is themost significant increase reported in the literature and further, is anotable improvement over the previously reported 12% increase after 14days in Irimeh et al., which also tested oral administration of fucoidanfrom Undaria pinnatifida. Importantly, Irimeh et al. reported 3 gram offucoidan administered daily, whereas the inventors achieved improvedresults using a 250 mg dosage regime. This highlights an important rolefor applying a specific dosage when orally administering fucoidan topromote release and circulation of CD34+ HSCs. Furthermore, a lowerdosage may permit longer-term patient use, such as routine dailyadministration, whereas higher dosages may not be compatible withrepeated and/or routine use.

Example 13 Decrease in CD34+HSCs Circulating in Peripheral BloodFollowing Oral Administration of Fucoidan from Chordaria cladosiphon

Extending these observations, the inventors discovered that fucoidanfrom several other algae species, including Chordaria cladosiphon,failed to elevate the circulating number of CD34+ HSCs in human subjects(FIG. 9). Despite application of several dosage regimes, including theeffective 250 mg dosage of fucoidan from Undaria pinnatifida asdescribed above, fucoidan from Chordaria cladosiphon resulted in adecrease in the number of circulating CD34+ HSCs, probably consequent toan increase in CXCR4 expression on the surface of circulating HSCs.These results reflect the complex interplay between the exact source offucoidan and identifying an effective therapeutic dose. Consumption of250 mg of this fucoidan from Chordaria cladosiphon gave an averagedecrease in the number of circulating stem cells (FIG. 3) using the samefucoidan preparation methods and administered under the same conditionsin volunteers, thereby demonstrating an effective role in supportingmigration of stem cells.

These results are consistent with earlier reports that fucoidan fromdifferent sources diverge in structure-activity relationships. Fucoidanfractions from A. nodosum and Pelvetia canculata have been reported topossess anti-coagulant activity through the tri-sulfated disaccharideheparin-like motif involved in HSC mobilization. Particularly notablewas the report that sulfation patterns correlated with theiranticoagulant activities. A similar molecule from the family ofgalactans, 3-linked, regularly 2-O-sulfated galactan, possessesanticoagulant activity not found in a corresponding 3-linked, regularly2-O-sulfated fucan. (Berteau and Mulloy, 2003; Mourão and Pereira, 1999;Pereira et al., 2002) These reports about anti-coagulant activity andthe inventors' observations about HSC mobilization clearly demonstratethat the structure-activity relationships of sulfated fucans, includingfucoidan, is not the result from generic features, such as chargedensity from the presence or absence of certain chemical groups.Instead, biological activity depends critically on the exact structureof the polysaccharide. Necessarily, the different structural fucoidansfrom distinct species of algae is expected to provide a complex range ofefficacies for various therapeutic applications, including HSCmobilization. As described above, this will also require establishingeffective therapeutic doses, which may vary when using fucoidans fromdifferent species.

Example 14 Various Methods of Preparing Polygonum multiflorum

A simple preparation of Polygonum multiflorum may be prepared byobtaining a portion of the fresh plant root that is then washed inwater, cleaned, cut into pieces, and then dried. For example, crushedroot may be diluted in water in a 12:1 extract as a simple preparation.

In another method of preparation, the Polygonum multiflorum may befrozen overnight at low temperatures (e.g., −70° C., −20° C., or 0° C.),and then crushed to form a solid powder, which can be dried at roomtemperature, or placed in a vacuum evaporator to remove moisture.

The solid powder may be immersed in water, an aqueous solution, or anorganic solvent to extract different components from Polygonummultiflorum. As described, certain high temperatures (e.g., boiling) oforganic solvents (e.g., ethanol or methanol) promote extraction ofcomponents, such as anthraquinones and derivatives, as they areotherwise insoluble in water, and room temperature organic solvents.Likewise, applying low temperature (4° C.) for extraction of aqueousfractions promotes stable isolation of certain components, such ashydroxyl stilbenes, as certain hydroxyl stilbenes degrade in a pH ortemperature dependent manner. Again, immersion in water, an aqueoussolution, or an organic solvent may be followed by drying at roomtemperature, or placed in a vacuum evaporator to remove moisture. Solidpowders may be lypophilized at various stages in order to aidsolubility.

Example 15 General Study Design for Polygonum multiflorum as a Stem CellMobilization Agent

Two consumables were tested in human subjects: Polygonum multiflorumextracts and a placebo. Peripheral venous blood samples were obtainedfrom healthy human volunteers between 20 and 45 years of age uponinformed consent. Blood and bone marrow samples were obtained underaseptic conditions and processed immediately. One gram of as 12:1preparation of Polygonum multiflorum extracts or placebo was given tovolunteers with 4-6 oz water. Appearance of the placebo was identical tothat of the Polygonum multiflorum extracts and consisted of tan-dyed,finely ground potato flakes encapsulated in vegetable capsules. Asdescribed in Example 10, the following exclusion criteria were used:under 20 or over 65 years of age, pregnancy, severe asthma and allergiesrequiring daily medication, any known chronic illness orprevious/current venereal disease, frequent recreational drug use, andimpaired digestive function (including previous major gastrointestinalsurgery). Seven volunteers were scheduled on two study days one weekapart. Testing was always performed at the same time of the day (8-11a.m.) to minimize the effect of circadian fluctuations. Volunteers wereinstructed to remain quiescent for 4 h, comfortably seated in a chair.After the first hour, the baseline blood sample was drawn. Immediatelyafter drawing the baseline sample, a consumable was provided. Bloodsamples were later drawn 60 and 120 min after ingestion of theconsumable. At each time point, 5 ml of blood was drawn into heparin,and 2 ml blood was drawn into EDTA. The blood vials were placed on arocking plate until use.

Example 16 Stem Cells Mobilize Following Consumption of Polygonummultiflorum Alone, or when Included in a Blend of Polygonum multiflorum,Lycium barbarum, Fucoidan, Colostrum, Spirulina and Mushroom

Polygonum multiflorum was shown to trigger a modest increase in thenumber of circulating stem cells by 13±6% (n=7) (p<0.05). The increaseexceeded 25% in 2 or the participants. The results are shown in FIG. 10.

Example 17 Stem Cells Mobilize Following Consumption of a Blend ofPolygonum multiflorum, Lycium barbarum, Fucoidan, Colostrum, Spirulinaand Mushroom

In addition Various compositions containing stem cell mobilizationagents, including Polygonum multiflorum may be prepared, including thefollowing components listed in Table 2 or 3. Administration of thesecompositions results in stem cell migration and/or mobilization.

TABLE 2 Composition 5 Composition 6 Composition 7 Composition 6 mg/dosemg/dose mg/dose mg/dose Polygonum multiflorum 250 250 500 1,000 Lyciumbarbarum (Goji extract) 250 250 500 0 Blue-green algae 25 100 250 0(Aphanizomenon flos aquae (AFA)) Fucoidan 0 400 500 1,000 Colostrum(Fractionated) 50 150 125 300 Spirulina 75 150 125 300 Mushroom 250 500750 1,000 Hericium erinaceus 83 166 249 332 Ganoderma Lucidum 83 166 249332 Cordyceps Sinensis 83 166 249 332

TABLE 3 Composition 9 Composition 10 Composition 11 Composition 12mg/dose mg/dose mg/dose mg/dose Polygonum multiflorum 25 400 300 400Lycium barbarum (Goji extract) 0 0 0 0 Blue-green algae 585 115 200 585(Aphanizomenon flos aquae (AFA)) Fucoidan 115 285 100 115 Colostrum(Fractionated) 0 50 100 0 Spirulina 0 50 100 0 Mushroom 375 200 450 0Hericium erinaceus 0 66 150 0 Ganoderma Lucidum 0 66 150 0 CordycepsSinensis 375 66 150 0

The various methods and techniques described above provide a number ofways to carry out the invention. Of course, it is to be understood thatnot necessarily all objectives or advantages described may be achievedin accordance with any particular embodiment described herein. Thus, forexample, those skilled in the art will recognize that the methods can beperformed in a manner that achieves or optimizes one advantage or groupof advantages as taught herein without necessarily achieving otherobjectives or advantages as may be taught or suggested herein. A varietyof advantageous and disadvantageous alternatives are mentioned herein.It is to be understood that some preferred embodiments specificallyinclude one, another, or several advantageous features, while othersspecifically exclude one, another, or several disadvantageous features,while still others specifically mitigate a present disadvantageousfeature by inclusion of one, another, or several advantageous features.

Furthermore, the skilled artisan will recognize the applicability ofvarious features from different embodiments. Similarly, the variouselements, features and steps discussed above, as well as other knownequivalents for each such element, feature or step, can be mixed andmatched by one of ordinary skill in this art to perform methods inaccordance with principles described herein. Among the various elements,features, and steps some will be specifically included and othersspecifically excluded in diverse embodiments.

Although the invention has been disclosed in the context of certainembodiments and examples, it will be understood by those skilled in theart that the embodiments of the invention extend beyond the specificallydisclosed embodiments to other alternative embodiments and/or uses andmodifications and equivalents thereof.

Many variations and alternative elements have been disclosed inembodiments of the present invention. Still further variations andalternate elements will be apparent to one of skill in the art. Amongthese variations, without limitation, are the sources of stem cellmobilization agents, the methods of preparing, isolating, or purifyingstem cell mobilization agents, analogs and derivatives thereof, methodsof treating various disease and/or conditions using stem cellmobilization agents, analogs and derivatives thereof, techniques andcomposition and use of solutions used therein, and the particular use ofthe products created through the teachings of the invention. Variousembodiments of the invention can specifically include or exclude any ofthese variations or elements.

In some embodiments, the numbers expressing quantities of ingredients,properties such as concentration, reaction conditions, and so forth,used to describe and claim certain embodiments of the invention are tobe understood as being modified in some instances by the term “about.”Accordingly, in some embodiments, the numerical parameters set forth inthe written description and attached claims are approximations that canvary depending upon the desired properties sought to be obtained by aparticular embodiment. In some embodiments, the numerical parametersshould be construed in light of the number of reported significantdigits and by applying ordinary rounding techniques. Notwithstandingthat the numerical ranges and parameters setting forth the broad scopeof some embodiments of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspracticable. The numerical values presented in some embodiments of theinvention may contain certain errors necessarily resulting from thestandard deviation found in their respective testing measurements.

In some embodiments, the terms “a” and “an” and “the” and similarreferences used in the context of describing a particular embodiment ofthe invention (especially in the context of certain of the followingclaims) can be construed to cover both the singular and the plural. Therecitation of ranges of values herein is merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g. “such as”) provided with respectto certain embodiments herein is intended merely to better illuminatethe invention and does not pose a limitation on the scope of theinvention otherwise claimed. No language in the specification should beconstrued as indicating any non-claimed element essential to thepractice of the invention.

Groupings of alternative elements or embodiments of the inventiondisclosed herein are not to be construed as limitations. Each groupmember can be referred to and claimed individually or in any combinationwith other members of the group or other elements found herein. One ormore members of a group can be included in, or deleted from, a group forreasons of convenience and/or patentability. When any such inclusion ordeletion occurs, the specification is herein deemed to contain the groupas modified thus fulfilling the written description of all Markushgroups used in the appended claims.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventor for carrying out the invention.Variations on those preferred embodiments will become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Itis contemplated that skilled artisans can employ such variations asappropriate, and the invention can be practiced otherwise thanspecifically described herein. Accordingly, many embodiments of thisinvention include all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

Furthermore, numerous references have been made to patents and printedpublications throughout this specification. Each of the above citedreferences and printed publications are herein individually incorporatedby reference in their entirety.

In closing, it is to be understood that the embodiments of the inventiondisclosed herein are illustrative of the principles of the presentinvention. Other modifications that can be employed can be within thescope of the invention. Thus, by way of example, but not of limitation,alternative configurations of the present invention can be utilized inaccordance with the teachings herein. Accordingly, embodiments of thepresent invention are not limited to that precisely as shown anddescribed.

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The invention claimed is:
 1. A method of increasing stem cellmobilization in a human in need thereof consisting essentially of:administering a therapeutically effective amount of an extract ofPolygonum multiforum, an extract of Cordyceps Sinensis and isolatedfucoidan to said human in need thereof in an amount sufficient toincrease stem cell mobilization in the human in need thereof wherein thestem cell mobilization is increased in the human in need thereof.
 2. Themethod of claim 1, wherein the stem cell is a bone marrow-derived stemcell.
 3. The method of claim 1, wherein the stem cell is a hematopoieticstem cell.
 4. The method of claim 1, wherein the administering is via anoral administration.
 5. The method of claim 4, wherein the oraladministration is via a capsule or a pill.
 6. The method of claim 1,wherein the therapeutically effective amount of an extract of Polygonummultiforum, is 10-2000 mg in a single dose.
 7. The method of claim 1,wherein the therapeutically effective amount of isolated fucoidan is10-2000 mg in a single dose.
 8. The method of claim 1, wherein thetherapeutically effective amount of an extract of Cordyceps Sinensis is10-1000 mg in a single dose.
 9. The method of claim 1, wherein thetherapeutically effective amount of an extract of Polygonum multiforumis 10-2000 mg in a single dose, the therapeutically effective amount ofisolated fucoidan is 10-2000 mg in a single dose and the therapeuticallyeffective amount of an extract of Cordyceps Sinensis is 10-1000 mg in asingle dose.